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Jun 2019

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Assessing Self-interaction of Mammalian Nuclear Proteins by Co-immunoprecipitation
共免疫沉淀法评价哺乳动物核蛋白的自身相互作用   

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Abstract

Protein-protein interactions constitute the molecular foundations of virtually all biological processes. Co-immunoprecipitation (CoIP) experiments are probably the most widely used method to probe both heterotypic and homotypic protein-protein interactions. Recent advances in super-resolution microscopy have revealed that several nuclear proteins such as transcription factors are spatially distributed into local high-concentration clusters in mammalian cells, suggesting that many nuclear proteins self-interact. These observations have further underscored the need for orthogonal biochemical approaches for testing if self-association occurs, and if so, what the mechanisms are. Here, we describe a CoIP protocol specifically optimized to test self-association of endogenously tagged nuclear proteins (self-CoIP), and to evaluate the role of nucleic acids in such self-interaction. This protocol has proven reliable and robust in our hands, and it can be used to test both homotypic and heterotypic (CoIP) protein-protein interactions.

Keywords: Immunoprecipitation (免疫沉淀反应), IP (免疫沉淀), Co-immunoprecipitation (免疫共沉淀), CoIP (免疫共沉淀), Self-interaction (自身相互作用), Cas9-mediated endogenous tagging (Cas介导内源性标记)

Background

Proteins are the building blocks of all living organisms, where they serve disparate functions that include structural, enzymatic, signaling and regulatory roles. Proteins perform most of these tasks by interacting with other proteins, small molecules (e.g., hormones) and macromolecules (e.g., nucleic acids, carbohydrates and lipids), with a wide range of affinities. In addition to these heterotypic interactions, several proteins are capable of homo-oligomerization (Paris et al., 2003; Akoev et al., 2004; Baisamy et al., 2005; Hwang et al., 2011; Chen et al., 2014; Kang et al., 2017), and perturbation of self-assembly capabilities can lead to pathological outcomes (Bourdenx et al., 2017; Yuan et al., 2018; Castle et al., 2019; Loughlin and Wilce, 2019). Our research is mainly focused on eukaryotic transcription factors (TFs), proteins that initiate and regulate gene transcription. Thanks to recent advances in super-resolution and single-molecule imaging, we are now appreciating that several TFs are not homogenously distributed in the nucleus, but rather locally concentrated in clusters (also hubs or condensates), and in rapid exchange between them (Hansen et al., 2017; Boehning et al., 2018; Cho et al., 2018; Chong et al., 2018; Dufourt et al., 2018; Mir et al., 2018). These clusters can emerge and be largely maintained by direct protein-protein self-association (e.g., cohesin self-interaction [Cattoglio et al., 2019]) or by a combination of protein and nucleic acid interactions (e.g., RNA-mediated CTCF self-association [Saldaña-Meyer et al., 2014; Hansen et al., 2019]).

While a few computational tools exist that predict protein self-interaction potential (Liu et al., 2013; Li et al., 2017; Zhai et al., 2017; Wang et al., 2018), these are most likely blind to nucleic-acid mediated homo-oligomerization, and of course demand biochemical validation. Available assays to probe protein self-interactions in-vitro include size-exclusion chromatography, microchip self-interaction chromatography and biochemical fractionation after density gradient centrifugation, all of which require purified recombinant proteins (García et al., 2003; Yusufzai et al., 2004; Saldaña-Meyer et al., 2014). Yeast or bacterial two-hybrid systems are an alternative in vivo genetic approach (Yusufzai et al., 2004; Kang et al., 2017), but again they do not probe self-association in the native protein environment. Co-immunoprecipitation assays (CoIPs) are by far the most common way to test heterotypic protein-protein interactions occurring in the relevant cell type. A few genetic engineering options are available to also detect homotypic protein-protein interactions by CoIP (Figure 1). One possibility is to overexpress a tagged version of the protein of interest (POI) in a given cell line and distinguish it from the endogenous one by virtue of its bigger size (Figure 1A). After immunoprecipitation with an antibody against the tag epitope, a Western blot is performed using an antibody recognizing both the tagged and the wild type protein. The appearance of a wild type band in the immunoprecipitated sample is considered evidence of self-interaction (Pant et al., 2004; He et al., 2008; Kang et al., 2017). This approach has several drawbacks: it uses an exogenously expressed protein, which can alter its physiological behavior, and it cannot exclude that the observed wild type band is simply a degradation product of the tagged protein. To address the latter, a more robust approach is perhaps to transfect two constructs, each expressing the POI tagged with a distinct epitope (“TAG1” and “TAG2” in Figure 1B). In this case, if the TAG1-tagged protein also pulls down the TAG2-protein, it is safe to conclude that the POI does self-interact. Alternatively, in a mixed in-vivo and in-vitro approach, a cell lysate overexpressing the POI tagged with one epitope is incubated with a recombinant POI tagged with an alternative epitope (Saldaña-Meyer et al., 2014). This method has the advantage of allowing easy screening of truncated versions of the POI to identify the regions responsible for self-association, but again relies on overexpression and requires purified proteins. An imaging-based approach, alternative to CoIP, to assess self-interaction of exogenously expressed proteins is the bimolecular fluorescence complementation assay, where the POI is fused to one or the other half of a split fluorescent protein (e.g., GFP or YFP) (Miller et al., 2015). Only when the protein self-associates is the fluorescent protein reconstituted and emits fluorescence (Kang et al., 2017). This assay does not overcome the overexpression concerns, and it is additionally limited by the irreversibility of the fluorescence reconstitution reaction, which can introduce biases and makes the method blind to the dynamics of the self-interaction.


Figure 1. Testing protein self-interaction by CoIP. Different strategies to genetically engineer cells and test the self-association of the POI by co-immunoprecipitation. A. A single plasmid encoding the tagged POI is transfected into cells in addition to the wild type protein expressed from the endogenous promoter (1). If the POI self-interacts, the wild type POI will associate both with itself (not shown) and with the exogenously expressed tagged version (2). A CoIP experiment that uses an antibody directed against the tag will also pull-down the wild type POI (3). If the tag is large enough, the tagged POI can be distinguished from the wild type one by size in a Western blot experiment using an antibody against the POI (4). B. Same as in (A) but this time two separate plasmids are transfected, each encoding the POI tagged with different epitopes (TAG1 and TAG2) (1). The immunoprecipitated material is subject to SDS-PAGE followed by Western blotting and self-interaction is assessed with antibodies against each tag (4). C. Our strategy to tag the endogenous POI with 2 different epitopes. A CRISPR/Cas9 targeting construct (for the expression of both Cas9 and the gRNA of choice) is transfected along with two donor plasmids, each containing the tag of choice (TAG1 or TAG2) flanked by genomic sequences homologous to the endogenous POI (LHR, left homology region, and RHR, right homology region) (1). Clones are selected and characterized that contain each endogenous allele tagged with one of the two epitopes (1’). If the POI self-interacts (2), CoIP experiments with antibodies against the TAG1 will also pulldown the TAG2-POI (3). The immunoprecipitated material is subject to SDS-PAGE followed by Western blotting and self-interaction is assessed using antibodies against each tag (4). Nucleases specific for either RNA (RNase), DNA (DNase) or both (benzonase) can be included in the experiment to test whether the self-association relies on protein-protein interactions and/or is mediated by nucleic acids. IN, input lysate; TAG IP, immunoprecipitated material.

Our approach to test self-association of TFs or other nuclear proteins expressed at physiological concentrations is to edit the genome of the cell line of interest via CRISPR/Cas9 (Reference 1; Roberts et al., 2017; Aird et al., 2018; Haupt et al., 2018; Sharma et al., 2018; Tasan et al., 2018; Zhang et al., 2018; Liu et al., 2019) and introduce two distinct tags in each allele encoding that TF (typically FLAG-Halo and V5-SNAPf) (Cattoglio et al., 2019; Hansen et al., 2019) (Figure 1C). The dual tagging can be attempted by providing 2 separate homology repair constructs together in parallel with CRISPR/Cas9, but we generally have higher success rate tagging each allele sequentially in a separate experiment, by using heterozygous clones from the first tagging and re-targeting the “wild type” allele (in fact, this is often a pseudo-wt allele, where Cas9 introduced indels at the cut site; this can be exploited to design another set of guide RNAs that will specifically target the untagged allele). Using the dual-tagged alleles we can then probe whether the TF self-interacts by co-immunoprecipitation (self-CoIP), and further investigate whether such self-association is mediated by protein-protein interactions and/or nucleic acids performing the experiment with or without specific nucleases. We provide here detailed instructions to perform the self-CoIP, while you can refer to our previous publications for the endogenous dual tagging strategy (Cattoglio et al., 2019; Hansen et al., 2019). This method cannot distinguish between a direct and an indirect self-interaction, and because it requires cell lysis and long incubation times, it cannot accurately estimate the amount of self-association happening in live cells, but rather give lower bounds. Nevertheless, endogenous tagging avoids artifacts originating from overexpression (Hansen et al., 2017; Shao et al., 2018), and, provided that all the deployed antibodies are specific (see Note 1), the self-CoIP method detailed here only requires standard lab equipment and reagents, and it has been working robustly and reproducibly in our hands with several TFs. While this protocol reliably detects self-association of nuclear proteins, we routinely use it to also test heterotypic protein-protein interaction in a standard CoIP setting, both in the nucleus and in the cytoplasm (see Note 2).

Materials and Reagents

  1. Pipette tips
  2. 150-mm TC-treated culture dishes (CorningTM 430599, Thermo Fisher Scientific, catalog number: 08-772-24)
  3. Cell scraper (Thermo Fisher Scientific, FisherbrandTM, catalog number: 08-100-241)
  4. Cuvettes (Thermo Fisher Scientific, FisherbrandTM, catalog number: 14-955-127)
  5. MagneSphere® technology twelve position, 1.5-ml magnetic separation stand (Promega, catalog number: Z5342)
  6. Empty gel cassettes, mini, 1.0 mm (Thermo Fisher Scientific, InvitrogenTM, catalog number: NC2010)
  7. Empty gel cassette combs, mini, 1.0 mm, 10 well (Thermo Fisher Scientific, InvitrogenTM, catalog number: NC3010)
  8. AmershamTM Protran® Western blotting membrane (Millipore Sigma, catalog number: GE10600041), Store at room temperature. Shelf-life: specified by manufacturer)
  9. 3 MM Blotting paper (Whatman, catalog number 3030-917)
  10. FalconTM Round-bottom polypropylene 5-ml tubes (CorningTM 352063, Thermo Fisher Scientific, catalog number: 14-959-11A)
  11. 50-ml conical tube (CorningTM 430291, Thermo Fisher Scientific, catalog number: 05-538-55A)
  12. Razor blades (VWR®, catalog number: 55411-050)
  13. Steriflip-GP sterile centrifuge tube top filter (Millipore Sigma, catalog number: SCGP00525)
  14. 10-ml syringe, Luer-LockTM (Becton Dickinson, catalog number: 302995)
  15. 22G PrecisionGlideTM needle (Becton Dickinson, catalog number: 305156)
  16. 1.5-ml low retention microcentrifuge tube (Phenix, catalog number: MH-815S)
  17. 12 x 24-mm round-bottom glass tube (Covaris®, catalog number: 520056)
  18. Disposable sterile bottle-top filters with 0.22 μm membrane (CorningTM 430513–500 ml or 430626–150 ml, Thermo Fisher Scientific, catalog number: 09-761-52 or 09-761-56)
  19. CL-XPosure film (Thermo Fisher Scientific, catalog number: 34091)
  20. Antibodies for the illustrative experiment detailed below:
    a. Mouse normal IgGs (Abcam, catalog number: ab37355)
    b. Mouse αFLAG (Millipore Sigma, catalog number: F3165)
  21. HEPES (Thermo Fisher Scientific, Fisher BioReagents, catalog number: BP310500), store at room temperature. Shelf-life: specified by manufacturer
  22. Hydrochloric acid (HCl, Thermo Fisher Scientific, catalog number: A144-500)
  23. Potassium chloride (KCl, Thermo Fisher Scientific, Fisher Chemical, catalog number: P217-10), store at room temperature. Shelf-life: specified by manufacturer
  24. Potassium hydroxide (KOH, Thermo Fisher Scientific, Fisher Chemical, catalog number: P250-500), store at room temperature. Shelf-life: specified by manufacturer
  25. Sodium hydroxide pellets (NaOH, Thermo Fisher Scientific, Fisher Chemical, catalog number: S318-100), store at room temperature. Shelf-life: specified by manufacturer
  26. Magnesium chloride hexahydrate (MgCl2·6H2O, Thermo Fisher Scientific, Fisher Chemical, catalog number: M33-500), store at room temperature. Shelf-life: specified by manufacturer
  27. Sodium chloride (NaCl, Thermo Fisher Scientific, catalog number: S271-10), store at room temperature. Shelf-life: specified by manufacturer
  28. 2-Propanol (Thermo Fisher Scientific, catalog number: A451-4), store at room temperature. Shelf-life: specified by manufacturer
  29. D-Sucrose (C12H22O11, Thermo Fisher Scientific, Fisher BioReagents, catalog number: BP220-212), store at room temperature. Shelf-life: specified by manufacturer
  30. Glycerol (C3H8O3, Thermo Fisher Scientific, Fisher Chemical, catalog number: G33-20), store at room temperature. Shelf-life: specified by manufacturer
  31. DTT (C4H10O2S2, GOLDBIO, catalog number: DTT10), storage temperature: -20 °C. Shelf-life: specified by manufacturer
  32. cOmpleteTM, EDTA-free protease inhibitor cocktail (Millipore Sigma, Roche, catalog number: 11873580001), storage temperature: 4 °C (tablets), -20 °C (dissolved, see Recipes)
  33. TritonTM X-100 (Millipore Sigma, catalog number: T9284), store at room temperature. Shelf-life: specified by manufacturer
  34. Aprotinin (Millipore Sigma, catalog number: A6279), storage temperature: 4 °C. Shelf-life: specified by manufacturer
  35. 20 mg/ml Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S), storage temperature: -20 °C. Shelf-life: specified by manufacturer. Use to create a BSA standard for protein quantification
  36. BSA (Millipore Sigma, catalog number: A7906), storage temperature: 4 °C. Shelf-life: specified by manufacturer. Use for preclearing magnetic beads.
  37. Benzamidine (Millipore Sigma, catalog number: B6506), storage temperature: 4 °C (powder), -20 °C (solution, see Recipes). Shelf-life: specified by manufacturer
  38. PMSF (Millipore Sigma, Roche, catalog number: 11359061001), storage temperature: room temperature. Shelf-life: specified by manufacturer
  39. EDTA (C10H18N2Na2O10, Thermo Fisher Scientific, Fisher Chemical, catalog number: S311-500), storage temperature: room temperature. Shelf-life: specified by manufacturer
  40. NP-40 alternative, protein grade detergent, 10% solution (Millipore Sigma, catalog number: 492018), storage temperature: 4 °C. Shelf-life: specified by manufacturer
  41. Benzonase® nuclease HC (Millipore Sigma, Novagen, catalog number: 71205-3), storage temperature: -20 °C. Shelf-life: specified by manufacturer
  42. Bio-Rad protein assay dye reagent (Bio-Rad, catalog number: 5000006), storage temperature: 4 °C. Shelf-life: specified by manufacturer
  43. DynabeadsTM Protein A or G (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10008D or 10009D), storage temperature: 4 °C. Shelf-life: 24 months from date of manufacture
  44. Precision plus proteinTM all blue prestained protein standards (Bio-Rad, catalog number: 1610393)
  45. MOPS (C7H15NO4S, Thermo Fisher Scientific, Fisher BioReagentsTM, catalog number: BP308-500), store at room temperature. Shelf-life: specified by manufacturer
  46. Tris base (C4H11NO3, Thermo Fisher Scientific, Fisher BioReagentsTM, catalog number: BP152-5), store at room temperature. Shelf-life: specified by manufacturer
  47. SDS (Bio-Rad, catalog number: 1610301), store at room temperature. Shelf-life: specified by manufacturer
  48. Ethanol (Decon Labs, Koptec pure ethanol–190 Proof, catalog number V1101)
  49. Bis-Tris (C8H19NO5, Millipore Sigma, catalog number: B9757), store at room temperature. Shelf-life: specified by manufacturer
  50. Sodium metabisulfite (Na2S2O5, Millipore Sigma, catalog number: S9000), Store at room temperature. Shelf-life: specified by manufacturer
  51. TEMED (Bio-Rad, catalog number: 1610800), storage temperature: 4 °C. Shelf-life: specified by manufacturer
  52. Ammonium persulfate (APS) (Bio-Rad, catalog number: 1610700), storage temperature: room temperature (powder), -20 °C (solution, see Recipes). Shelf-life: specified by manufacturer (powder), 6 months or more (solution)
  53. ProtoGel (30%) 37.5:1 Acrylamide to Bisacrylamide solution (national diagnostics, catalog number: EC-890), storage temperature: 4 °C. Shelf-life: specified by manufacturer
  54. UltrapureTM water (Thermo Fisher Scientific, Fisher BioReagentsTM, catalog number: 10977023), store at room temperature. Shelf-life: specified by manufacturer
  55. Glycine (C2H5NO2, Thermo Fisher Scientific, Fisher BioReagentsTM, catalog number: BP381-5), store at room temperature. Shelf-life: specified by manufacturer
  56. Methanol (CH4O, Thermo Fisher Scientific, Fisher Chemical, catalog number: A452-4), store at room temperature. Shelf-life: specified by manufacturer
  57. Tween®-20 (Millipore Sigma, Calbiochem, catalog number: 655205), store at room temperature. Shelf-life: specified by manufacturer
  58. Instant Nonfat dry milk, pasteurized extra grade (Signature Kitchens), store at room temperature. Shelf-life: specified by manufacturer
  59. 2-mercaptoethanol (Millipore Sigma, catalog number: M3148), store at room temperature. Shelf-life: specified by manufacturer
  60. Bromophenol blue sodium salt (C19H9Br4NaO5S, Millipore Sigma, catalog number: B5525), Store at room temperature. Shelf-life: specified by manufacturer
  61. Potassium phosphate monobasic (Na2HPO4, Thermo Fisher Scientific, Fisher Chemical, catalog number: P285-500)
  62. Sodium phosphate dibasic anhydrous (KH2PO4, Thermo Fisher Scientific, Fisher Chemical, catalog number: S375-212)
  63. Western lightning Plus-ECL (PerkinElmer, catalog number: NEL103E001EA), storage temperature: 4 °C. Shelf-life: specified by manufacturer
  64. 70% ethanol
  65. 6 N HCl (see Recipes)
  66. 10x PBS (see Recipes)
  67. 1x PBS, pH 7.4 (see Recipes)
  68. 1 M HEPES (see Recipes)
  69. 1 M Tris-base pH 6.8/7.5 (see Recipes)
  70. 5 M NaCl (see Recipes)
  71. 2 M KCl (see Recipes)
  72. 1 M MgCl2 (see Recipes)
  73. 1 M DTT (see Recipes)
  74. 10% Triton X-100 (see Recipes)
  75. 0.5 M EDTA pH 8.0 (see Recipes)
  76. 10% APS (see Recipes)
  77. 50x cOmplete protease inhibitors stock (see Recipes)
  78. 1 M benzamidine (see Recipes)
  79. 0.2 M PMSF (see Recipes)
  80. 1.23 M Bis-Tris pH 6.4/6.7 (see Recipes)
  81. Cell lysis buffer (see Recipes)
  82. 0.1 M CoIP buffer (see Recipes)
  83. 0.2 M CoIP buffer (see Recipes)
  84. 0.5% BSA in 0.2 M CoIP buffer (see Recipes)
  85. 20x MOPS running buffer (see Recipes)
  86. Transfer buffer (see Recipes)
  87. TBS-T (see Recipes)
  88. 4x sample buffer (see Recipes)
  89. Blocking solution (see Recipes)
  90. Blotting solution (see Recipes)

Equipment

  1. -80 °C freezer
  2. Pipettes (P20, P200, P1000)
  3. DynaMagTM-15 magnet (Thermo Fisher Scientific, catalog number: 12301D)
  4. Focused-ultrasonicator (Covaris®, model: S220) with 12 x 24-tube adaptor (Covaris®, catalog number: 500199)
    Note: You can replace this instrument with any type of sonicator (e.g., ultrasonic processors). We recommend sonication to solubilize chromatin in the absence of nuclease digestion. You can skip the sonication steps when working with cytosolic proteins (note on Step C5) or if digesting with nuclease the entire sample.
  5. Refrigerated table-top centrifuge (Eppendorf, model: 5810R)
  6. Refrigerated microfuge (Eppendorf, model: 5418R)
  7. UV-VIS Spectrophotometer (Shimadzu, model: UV-1900)
  8. FisherbrandTM IsotempTM Digital Block Heater (Thermo Fisher Scientific, catalog number: 88-860-021)
  9. XCell SureLockTM Mini-Cell (Thermo Fisher Scientific, catalog number: EI0001)
  10. Electrophoresis power supply (Thermo Fisher Scientific, model: EC-105); use for SDS-PAGE
  11. Mini Trans-Blot® electrophoretic transfer cell (Bio-Rad, catalog number: 1703930)
  12. PowePacTM Basic Power supply (Bio-Rad, catalog number: 1645050); use for transfer
  13. Clay Adams nutator mixer (Becton Dickinson); use to rock lysates
  14. Rocker platform (Bellco Biotechnology, model: 7740-10010): use to blot Western blot membranes with antibodies overnight
  15. Benchtop orbital shaker (Thermo Fisher Scientific, model: MaxQTM 2000)

Procedure

  1. Collect cells
    1. Grow your cell line of interest to 80% confluency.
      Note: For mouse embryonic stem cells, we typically start with two 150-mm confluent tissue culture plates (~30 x 106-40 x 106 total cells). The amount of plates to use will depend on how many cells you can obtain from each plate (e.g., mouse embryonic stem cells grow in 3D colonies and thus give a very good yield) and on the cellular abundance of the protein to be immunoprecipitated.
    2. For adherent cells:
      1. Aspirate media.
      2. Wash twice with ice-cold 1x PBS:
        1. Pour enough PBS onto the plate to cover it and swirl it around to remove media residues.
        2. Aspirate the PBS or simply invert the plate to discard it.
      3. Transfer the plate(s) on ice.
        Note: From now on, keep samples and buffers in ice all the time, unless otherwise specified.
      4. Scrape cells with ice-cold 1x PBS added of PMSF, benzamidine and aprotinin (use protease inhibitors’ stock solutions as 1,000x). We typically use 3-4 ml of PBS per 150-mm plate.
      5. Transfer scraped cells to 15 ml conical tubes (or 50 ml if the final volume exceeds 12 ml).
      6. Collect all the remaining cells by washing the scraped plate(s) with another 3-4 ml of 1x PBS with protease inhibitors and transfer to the same 15 ml conical tube(s).
      7. Pellet cells in a refrigerated table-top centrifuge at 4 °C and 300 x g for 10 min.
      8. Aspirate the supernatant and flash-freeze cell pellets in liquid nitrogen and store at -80 °C until ready to proceed.
    3. For cells growing in suspension:
      1. Transfer cells from the culture vessel to a 50 ml conical tube; wash the culture vessel once with 1x PBS to collect all the remaining cells.
      2. Pellet cells in a table-top centrifuge at 25 °C and 300 x g for 5 min.
      3. Transfer the cell pellet on ice.
      4. Wash once with 40 ml of ice-cold 1x PBS:
        1. Resuspend the pellet in PBS.
        2. Pellet in a refrigerated table-top centrifuge at 4 °C and 300 x g for 10 min.
      5. Resuspend the pellet in 20 ml of ice-cold 1x PBS added of PMSF, benzamidine and aprotinin.
      6. Pellet cells in a refrigerated table-top centrifuge at 4 °C and 300 x g for 10 min.
      7. Aspirate the supernatant and flash-freeze cell pellets in liquid nitrogen and store at -80 °C until ready to proceed.

  2. Turn on the Covaris sonicator and set at 4 °C
    Note: It takes ~30-45 min for the sonicator to complete degassing and cool down.

  3. Lyse nuclei (all volumes refer to 2x 150-mm plates, or 30-40 million cells)
    1. Prepare the cell lysis buffer (see Recipe B1; 700 μl for 2x 150-mm plates, or 30-40 million cells), adding protease inhibitors (PMSF, aprotinin, benzamidine, and the Roche complete inhibitors–50x stock) and Triton X-100 to 0.1% final concentration (use the 10% stock solution as 100x).
    2. Resuspend each pellet in 700 μl of the freshly prepared cell lysis buffer using a P1000 pipet.
    3. Transfer the lysate to low-retention 1.5-ml tubes and rock at 4 °C for 8 min to allow lysis (e.g., using a rotator in the cold room).
    4. Spin in a refrigerated microfuge at 4 °C and 3,000 x g for 3 min.
    5. Optional: keep the supernatant as the “cytoplasmic fraction”.
      Note: This protocol is specifically optimized for nuclear proteins, but you can also perform the self-CoIP on the cytoplasmic fraction.
    6. Remove the supernatant with a P1000 first, and then with a P200, without disturbing the nuclear pellet.
    7. Prepare the 0.1 M CoIP buffer (see Recipe B2; 700 μl for 2x 150-mm plates, or 30-40 million cells), adding protease inhibitors (PMSF, aprotinin, benzamidine, and the Roche complete inhibitors–50x stock).
    8. Resuspend each nuclear pellet in 700 μl of 0.1 M CoIP buffer.
      Note: If you are not interested in performing a nuclease digestion, resuspend the nuclear pellet directly in 0.2 M CoIP buffer, sonicate the lysate and jump from Steps C10 to C17.
    9. Transfer the resuspended nuclei to 12 x 24-mm Covaris tubes.
    10. Sonicate each sample (Peak power: 100 W, Cycles/burst: 200, Duty Factor: 20, 8 repeats of 20 seconds of treatment followed by a 40-s delay).
    11. Measure the samples’ volume while transferring them back to a 1.5-ml low retention tube.
    12. Bring the volume to 2 ml with 0.1 M CoIP buffer plus protease inhibitors.
    13. Equally divide the sample to two tubes, 1 ml to be left untreated and 1 ml to treat with Benzonase (1 μl per ml of lysate).
      Note: Benzonase digests both DNA and RNA, and thus will tell you whether the self-interaction is mostly mediated by protein-protein interactions or it requires nucleic acids. If the self-association is dependent on nucleic acids, you can test whether RNA or DNA are involved replacing benzonase with either RNaseI or DNaseI (you will need to adjust the digestion buffer for DNaseI).
    14. Rock at 4 °C for 3 to 4 h to achieve complete Benzonase digestion.
    15. Spin briefly in a refrigerated microfuge at 4 °C to pull liquid down.
    16. Add 5 M NaCl to bring the final salt concentration to 0.2 M (i.e., 20.4 μl per 1 ml of lysate) and immediately invert the tubes.
    17. Rock at 4 °C for 30 min to allow complete nuclear lysis.
    18. Spin in a refrigerated microfuge at 4 °C and at maximum speed for 20 min.
    19. Transfer supernatants to new low-retention 1.5-ml tubes and quantify by Bradford assay.

  4. Quantify nuclear lysates
    1. Turn on the UV/VIS spectrophotometer.
    2. Prepare enough Bradford reagent to quantify all your lysates plus 6 additional samples to prepare a BSA standard curve (blank, 2.5 μg, 5 μg, 7.5 μg, 12 μg). You will need 1 ml of Bradford per sample (dilute the Bio-Rad protein assay dye reagent 1:5 in ddH2O).
    3. Prepare your BSA standard, adding 47.5 μl of 0.2 M CoIP buffer to 2.5 μl of 20 mg/ml BSA (NEB). This will give you a 1 μg/μl BSA solution.
    4. Aliquot the Bradford reagent to 1.5-ml tubes as follows:
      1 ml (blank)
      997.5 μl Bradford + 2.5 μl BSA (2.5-μg standard)
      995 μl Bradford + 5 μl BSA (5-μg standard)
      992.5 μl Bradford + 7.5 μl BSA (7.5-μg standard)
      988 μl Bradford + 12 μl BSA (12-μg standard)
      998 μl Bradford + 2 μl sample #1/untreated
      998 μl Bradford + 2 μl sample #1/Benzonase
      For as many samples as you need to quantify.
    5. Mix by inverting each tube right after adding BSA/lysate.
    6. Pour the content of each tube into a plastic cuvette and read the absorbance at 595 nm (Auto Zero the instrument with the blank solution).
    7. Use the template provided in Supplemental Table S1 to build a standard curve with the BSA readings and to interpolate the concentration of your samples.

  5. Setup your self-CoIP experiment and pre-clear lysates and beads
    We are here illustrating a typical self-CoIP experiment to probe the self-association of the cohesin subunit Rad21, and its dependency on nucleic acids. Specifically, we are using nuclear lysates from mouse embryonic stem cells with an endogenously dually tagged Rad21 protein (the B4 clone originally described in Cattoglio et al. (2019). This cell line contains a Rad21-SNAPf-3xFLAG allele and a Rad21-Halo-V5 allele (Figure 2A). We will pulldown the protein expressed from the first allele with a FLAG antibody (Figure 2B) and check whether the V5-tagged protein produced from the second allele is also immunoprecipitated using a V5 antibody during the Western blot (Figure 2C), both in untreated lysates and in lysates treated with benzonase to digest nucleic acids. We will also check the total IP efficiency of the experiment blotting against the immunoprecipitated FLAG protein (Figure 2D).


    Figure 2. Self-CoIP of Rad21. Experimental workflow and results of the Rad21 self-CoIP assay used here to illustrate the protocol. The data in C and D is modified from the original Figure 2F in Cattoglio et al. (2019). See Procedure E for a detailed description. IN, input nuclear lysates; UT, untreated lysates; Benz: benzonase-treated lysates.

    1. Create a scheme for your experiment like the following:
      Sample # (mg needed)
      Sample 1: mESC/untreated_input (0.2 mg)
      Sample 2: mESC/untreated_IgG (1 mg)
      Sample 3: mESC/untreated_αFLAG (1 mg)
      Sample 4: mESC/benzonase_input (0.2 mg)
      Sample 5: mESC/benzonase_IgG (1 mg)
      Sample 6: mESC/benzonase_αFLAG (1 mg)
      In this case, you will need 2.2 mg of nuclear lysates per condition (untreated and benzonase-treated).
      Note: The normal IgG sample (immunoglobulins purified from pre-immune serum) controls for non-specific pulldown and it must be of the same species of your αFLAG antibody (e.g., mouse anti-FLAG antibody and normal mouse IgG).
    2. Dilute the amount of lysates needed for your experiment to 1 mg/ml with 0.2 M CoIP buffer added of protease inhibitors (PMSF, aprotinin, benzamidine and Roche cOmplete inhibitors). If your volume exceeds 1.5 ml, move the diluted lysates to round-bottom tubes (Figure 3a).
      In our illustrative experiment:
      Condition
      Concentration (Table S1)
      For 2.2 mg
      0.2 M CoIP buffer to 1 mg/ml
      mESC/untreated
      2.5 mg/ml
      880 μl
      1,320 μl
      mESC/benzonase
      2.8 mg/ml
      786 μl
      1,414 μl


      Figure 3. Step-by-step self-CoIP protocol. The figure illustrates all the steps required to immunoprecipitate the protein of interest, described in detail in the text.

    3. Preclear lysates with beads to reduce non-specific binding (note that these beads will be discarded at the end of the preclearing) (Figure 3b).
      1. Wash protein A or G Dynabeads.
        Note: Choose either protein A or G depending on the species and IgG subtype of your primary antibody (e.g., use protein G for rabbit and mouse IgG2a antibodies and protein A for guinea pig). Check the specification sheet of the magnetic beads manufacturer to confirm your choice.
        1. Resuspend vigorously the Dynabeads until homogenously dispersed.
        2. For each condition, pipet 20 μl of Dynabeads per mg of lysate (e.g., 44 μl for 2.2 mg) to a low-retention 1.5 ml tube.
        3. Resuspend in 500 μl of 0.2 M CoIP buffer.
        4. Insert the tube in the magnet separation rack and wait for 5 min for the solution to clear.
        5. Aspirate the CoIP buffer with a pipet, remove the tube from the magnet and repeat the wash a second time (Steps E3a-iii to E3a-v).
      2. Remove the tube from the magnet and use the diluted lysate to resuspend the beads and transfer them to the round-bottom tubes with the rest of your nuclear proteins.
      3. Rock the lysates with the beads for at least 2 h at 4 °C.
    4. Preclear beads with BSA (note that you will use these beads to perform the actual pulldown experiment) (Figures 3e-3f)
      1. Wash protein A or G Dynabeads; you will prepare a single pool of Dynabeads to be used with all your samples.
        1. Resuspend vigorously the Dynabeads until homogenously dispersed.
        2. In 1.5-ml tubes, pipet 20 μl of Dynabeads per sample (e.g., 20 μl for 6 samples: 120 μl).
          Note: You will not pipet Dynabeads in your input samples, but we include inputs when preclearing beads to accommodate pipetting errors later on.
        3. Resuspend in 1 ml of 0.2 M CoIP buffer.
        4. Insert the tube in the 1.5-ml magnetic separation stand and wait for 5 min for the solution to clear.
        5. Aspirate the CoIP buffer with a pipet, remove the tube from the magnet and repeat the wash a second time (Steps E4a-iii to E4a-v).
      2. Remove the tube from the magnet and resuspend the beads in 0.2 M CoIP buffer with 0.5% BSA (see Recipe B4). Use an amount of buffer equivalent to 10 times the volume of your beads (e.g., 1,200 μl BSA buffer for 120 μl of beads).
      3. Rock the beads with BSA at 4 °C overnight.
        Note: For convenience, we preclear beads with BSA overnight while incubating the lysates with antibodies, but a couple of hours are also sufficient.

  6. Add antibodies to the precleared lysates (Figures 3c-3d).
    1. Briefly spin down the precleared lysates at 4 °C to remove liquid drops from the tubes’ lids.
    2. Remove the snap cap and insert the tubes in the DynaMagTM magnetic stand.
    3. Wait for 5 min for the solution to clear .
      Note: During this and all subsequent magnetic separations, we like to transfer the magnetic stand in the fridge while waiting for the solution to clear, to maintain samples cold at all times.
    4. Transfer the supernatant to a new tube without disrupting the beads.
    5. Prepare as many 1.5-ml low-retention tubes as needed for the IP (e.g., for our 6 samples).
    6. Distribute the precleared lysates to the new tubes. In our illustrative experiments this will correspond to:
      Samples 2-3 and 5-6: pipet 1 ml (corresponding to 1 mg)
      Samples 1 and 4 (inputs): pipet 100-150 μl (corresponding to 0.1-1.15 mg)
    7. Add the specific antibody (4 μg per mg of lysate) and immediately invert the tube by mixing. In our illustrative experiment:
      Sample 1 (mESC/untreated_input ): no antibody
      Sample 2 (mESC/untreated_IgG): 4 μg of mouse normal IgGs
      Sample 3 (mESC/untreated_αFLAG): 4 μg of mouse αFLAG
      Sample 4 (mESC/benzonase_input): no antibody
      Sample 5 (mESC/benzonase_IgG): 4 μg of mouse normal IgGs
      Sample 6 (mESC/benzonase_αFLAG): 4 μg of mouse αFLAG
    8. Rotate/rock the lysates with antibodies at 4 °C overnight.
      Note: We obtain best results when incubating the antibody overnight, but you can test your antibody and try to shorten the time.

  7. Bind antibodies to the precleared magnetic beads (Figures 3g-3i).
    1. Retrieve the samples and the BSA-precleared beads and briefly spin them down at 4 °C to remove liquid drops from the tubes’ lids.
    2. Place the tube with precleared beads in the magnetic separator rack.
    3. Wait for 5 min for the solution to clear .
    4. Remove the tube from the magnet and resuspend beads in 0.2 M CoIP buffer with inhibitors (50 μl per sample; in our illustrative experiment, 50 μl x 6 samples = 300 μl total).
    5. Distibute 50 μl of resuspended beads to each sample to be immunoprecipitated (inputs excluded) and immediately invert to mix.
    6. Rotate/rock the lysates with antibodies and beads at 4 °C for 2 h minimum.
    7. Leave input samples on ice.

  8. Wash immunoprecipitated samples (IP samples) to remove non-specific binding (Figure 3j).
    1. Prepare enough ice-cold 0.2 M CoIP buffer plus protease inhibitors to wash each IP sample 8 times with 400 μl of buffer (e.g., 4 IP samples x 400 μl x 8 = 12.8 ml of 0.2 M CoIP buffer; inputs are excluded).
    2. Briefly spin down IP samples at 4 °C to remove liquid drops from the tubes’ lids.
    3. Insert the IP samples’ tubes in the 1.5-ml magnetic stand.
    4. Wait for 5 min for the solution to clear . At this point your immunoprecipitated protein is attached to the beads via the antibody.
    5. Remove the CoIP buffer with a P1000 pipet without disturbing the beads.
    6. Remove tubes from the magnet and put them back on ice.
    7. Add 400 μl of 0.2 M CoIP buffer.
      Note: Make sure that the beads are completely resuspended. We typically vortex at low speed.
    8. Repeat Steps H4-H7 for 8 washes total.
    9. After the last wash, also remove any residual buffer with a P20.

  9. Elute immunoprecipitated material and prepare inputs and samples for SDS-PAGE (Figures 3k-3l).
    1. Remove tubes from the magnet and put them back on ice.
    2. Elute immunoprecipitated proteins from the beads adding 20 μl of 1x SDS loading buffer (Recipe B8).
      Note: The elution volume depends on how many samples you have. We are here illustrating an experiment that uses a 10-well acrylamide gel, which can easily accommodate 17 μl. We also use 12- or 15-well gels, and adjust the elution volumes accordingly or load less material.
    3. Vortex to resuspend.
    4. Boil the beads in a thermal block set at 98 °C for 5 min.
    5. Spin briefly.
    6. Place tubes in the 1.5-ml magnetic stand.
    7. Wait for 5 min. Your eluted proteins are now in the supernatant.
    8. Transfer the supernatant to a new low-retention 1.5 ml tube on ice (the volume should be slightly more than 20 μl). You will load 17 μl of this undiluted sample on one gel to estimate the self-CoIP efficiency (Figure 2C).
    9. Prepare a new set of tubes with 18 μl of 1x SDS-loading buffer and add 2 μl of the eluted material. You will load 17 μl of this 1:10 dilution on a second gel to estimate the IP efficiency (Figure 2D).
    10. Prepare 0.75% inputs to load along with your IP samples.
      1. Mix 22 μl of input lysate with 25 μl of 2x SDS-loading buffer and 3 μl of 0.2 M CoIP buffer.
        Note: This dilutes your inpute lysate to 0.44 μg/μl. Loading 17 μl of this dilution will give you ~7.5 μg, which correspond to 0.75% of the starting material (1 mg).
      2. Boil 5 min in a thermal block set at 98 °C.
      3. Spin briefly and place tubes on ice.

  10. Evaluate self-CoIP and CoIP efficiency by SDS-PAGE and Western blotting
    1. Prepare SDS-PAGE acrylamide gels
      Note: If more convenient, you can purchase pre-cast NuPAGE Bis-Tris gels from Invitrogen.
      1. Choose a gel percentage depending on the size of your POI. Gel migration charts are available on the ThermoFisher Scientific website (our gel recipe behaves like the NuPAGE Bis-Tris pre-casted gels form InvitrogenTM) (Reference 33). For Rad21 we use 9% gels.
      2. Check Table 1 for Recipes.

        Table 1. Use this table to prepare your SDS-PAGE gel (all specified volumes are for 1-mm cassettes, 1 gel)
        Separating gel
        Reagent 12% 10% 9.5% 9% 8%
        1.23 M Bis-Tris pH 6.7 1.8 ml 1.8 ml 1.8 ml 1.8 ml 1.8 ml
        30% Acrylamide 2.52 ml 2.1 ml 2.0 ml 1.89 ml 1.68 ml
        double-distilled H2O 1.93 ml 2.35 ml 2.45 ml 2.56 ml 2.77 ml
        10% APS 42 µl 42 µl 42 µl 42 µl 42 µl
        TEMED 12 µl 12 µl 12 µl 12 µl 12 µl
        Total volume: 6.3 ml 6.3 ml 6.3 ml 6.3 ml 6.3 ml
        Stacking gel
        Reagent 4%
        1.23 M Bis-Tris pH 6.4 710 µl
        30% Acrylamide 330 µl
        double-distilled H2O 1.43 ml
        10% APS 20 µl
        TEMED 7 µl
        Total volume: 2.5 ml

      3. Place two 1-mm gel cassettes in a stable upright position.
      4. In a 15 ml conical tube, start preparing the separating gel, adding reagents in the order specified in Table 1 (Bis-Tris, acrylamide and water).
      5. Mix well pipetting up and down and finally add APS and TEMED.
      6. Prompty mix and pipet into the gel cassettes, leaving enough space for the stacking gel.
      7. Immediately layer 70% ethanol on top of the gel, using a squirt bottle. This will make your gel front neat and straight.
      8. Wait for polymerization to complete.
        Note: When solidified, the gel front will appear as a neat line easily distinguishable from the ethanol layered above.
      9. Aspirate the ethanol and wash the gel extensively with ddH2O using a squirt bottle.
      10. Aspirate the excess ddH2O.
      11. In a new 15 ml conical tube, start preparing the stacking gel, adding reagents in the order specified in Table 1 (Bis-Tris, acrylamide and water).
      12. Mix well pipetting up and down and finally add APS and TEMED.
      13. Prompty mix and pipet into the gel cassette on top of the separating gel, all the way to the top of the cassette.
      14. Slowly place the selected combs (in our case 10-well combs), making sure no bubbles form below the wells.
        Note: We normally aspirate the excess stacking gel with a P1000 while inserting the comb, transferring it back to the 15 ml tube where we prepared it, so that no acrylamide gets spilled on the bench.
      15. Wait for polymerization to complete.
    2. Run the SDS-PAGE
      1. Transfer the gels into a running cassette (each cassette can accommodate 2 gels) and lock them in place.
        Note: Remember to peel off the white adhesive at the bottom of the gel cassette.
      2. Dilute the 20x MOPS running buffer according to Recipe B5.
        Note: Remember to add sodium metabisulfite.
      3. Remove the combs from the gel cassettes.
      4. Fill the inside chamber between the two cassettes with running buffer all the way to the top.
        Note: This will allow you to check whether the cassettes are properly locked or rather the buffer is leaking.
      5. Gently wash the wells using a syringe with a 22G needle filled with running buffer. Do not disturbe the bottom of the wells.
      6. Add the rest of the running buffer.
        Note: 500 ml will fill half of the outer tank or less.
      7. Decide the loading order of your samples. In our illustrative experiment:

        Gel#1 (αV5 blot, to evaluate self-CoIP)
        1)
        empty
        2)
        Prestained protein standard
        3)
        mESC/untreated_0.75%_input
        4)
        mESC/untreated_IgG_undiluted
        5)
        mESC/untreated_ αFLAG _undiluted
        6)
        empty
        7)
        mESC/benzonase_0.75%_input
        8)
        mESC/benzonase_IgG_undiluted
        9)
        mESC/benzonase_ αFLAG _undiluted
        10)
        empty

        Gel#2 (αFLAG blot, to evaluate CoIP)
        1)
        Prestained protein standard
        2)
        empty
        3)
        mESC/untreated_0.75%_input
        4)
        mESC/untreated_IgG_diluted
        5)
        mESC/untreated_ αFLAG _diluted
        6)
        empty
        7)
        mESC/benzonase_0.75%_input
        8)
        mESC/benzonase_IgG_diluted
        9)
        mESC/benzonase_ αFLAG _diluted
        10)
        empty
        Note: The protein standard is either loaded in well 1 (gel#1) or well 2 (gel#2) so that the two can be easily distinguished.
      8. Prepare the prestained protein standard (7 μl plus 27 μl of 1x SDS-loading buffer, 17 μl per well).
      9. Carefully load 17 μl of each sample with a P20. Fill the empty wells with 17 μl of 1x SDS loading buffer.
      10. Connect the lid of the running cassette to a power supply and place it on top of the cassette.
      11. Run at 75 V until the samples enter the separating gel.
        Note: Once the marker hits the separating gel, you will see clear bands forming.
      12. Increase the voltage to 130 V.
        Note: To help heat dissipation, we often put the cassette on top of a large metal block.
      13. Run until the bromophenol blue hits the bottom of the cassette, but before it runs out of the gel.
      14. Turn off the power supply.
      15. Remove the lid, unlock the gel cassettes and take them out.
      16. Wash extensively the cassettes with ddH2O, including the wells, to remove any traces of running buffer.
    3. Transfer proteins to a nitrocellulose membrane
      1. Assemble the transfer apparatus (Figure 4).
        1. Pre-wet with ddH2O two pieces of blotting membrane, cut only slightly larger than the gel to transfer.
          Note: Handle the membrane with gloves and only touching the membrane’s sides.
        2. Take a tray that will fit the gel holder cassettes.
        3. Add a gel holder cassette, black side facing down, and pour enough transfer buffer to cover it.
        4. Add one sponge, and make sure it gets fully covered in transfer buffer.
        5. Pre-wet in transfer buffer 2 pieces of blotting paper, cut only slightly larger than the gel to transfer.
        6. Place the first gel cassette on a paper towel (wells down) and crack it open with the provided metal spatula.
        7. Remove the top part, making sure that the gels adheres to the bottom one.
        8. Use a razor blade to remove the bottom part of the gel that sticks out, and the stacking gel.
          Note: The stacking gel is gluey. Scrape it with the razor blade from the gel cassette to the paper towel to get rid of all residues.
        9. Submerge the cassette containing the gel in the tray with transfer buffer.
        10. Lift the gel from the cassette and transfer it on top of the wet blotting paper; keep the empty cassette on the side.
        11. Pre-wet the blotting membrane in transfer buffer and layer it on top of the gel.
        12. Pre-wet a piece of blotting paper and layer it on top of the membrane.
        13. Use the empty gel cassette to roll out any air bubbles that might have formed between the gel and the membrane, pressing firmly but gently.
        14. Repeat Steps J3a-xii and J3a-xiii.
        15. Pre-wet another sponge and add it on top.
          Note: Always pre-wet blotting paper and sponges to avoid any capillarity effects.
        16. Carefully close the gel holder cassette without disturbing the sandwich and insert it in the transfer tank.
        17. Repeat Steps J3a-iii through J3a-xvi for the second gel.


          Figure 4. Assembly of the transfer “sandwich”

      2. Insert the cooling unit.
      3. Completely fill the transfer tank.
      4. Close the lid and connect the cables to the transfer power supply.
      5. Transfer at 100 V at 4 °C for 2 h.
    4. Block the membrane
      1. Retrieve the blotted membranes from the transfer apparatus.
        Note: You will know that the transfer was successful if you see the stained molecular weight transferred to your membrane.
      2. Quickly rinse each membrane in TBS-T using a container big enough to accommodate the membrane without bending it.
      3. Add 10 ml of blocking solution (10% milk in TBS-T).
        Note: Pipet liquid next to the membrane, never on top of it.
      4. Agitate for at least 1 h at room temperature.
    5. Blot the membrane
      1. Prepare your primary antibodies in blotting solution (5% milk in TBS-T) in 15 ml conical tubes. In our illustrative experiment:
        Gel#1: rabbit αV5, 1:2,000 (4 μl in 8 ml of 5% milk)
        Gel#2: rabbit αFLAG, 1:1,000 (8 μl in 8 ml of 5% milk)
        Note: Check the datasheet of your antibody of choice to find the recommended dilution for Western blot. This can vary a lot from one antibody to another.
      2. Discard the blocking solution and add the blotting solution with antibodies.
      3. Agitate at 4 °C overnight.
        Note: Overnight incubation in the cold room increases the specific signal while reducing background noise for all the antibodies we tested so far.
    6. Wash the membrane
      1. Remove the blotting solutions from the membrane.
      2. Remove any primary antibody residue with a quick rinse with 10 ml of TBS-T.
      3. Replace with fresh 10 ml of TBS-T and agitate for 15 min.
      4. Replace with fresh TBS-T and agitate for 5 min.
      5. Repeat Step J6d.
    7. Blot with secondary antibodies conjugated to HRP
      1. Prepare a 1:5,000 dilution of the HRP-cojugated secondary antibody in 5% milk in TBS-T. In our illustrative experiment, goat α-mouse HRP (2 μl in 10 ml of 5% milk).
      2. Agitate at room temperature for a minimum of 30 min.
    8. Wash the membrane as in Step J6.
    9. Perform the chemiluminescent reaction
      1. For each membrane, prepare 2 ml of ECL reagent (1 ml of the oxidiziong reagent plus 1 ml of the enhanced luminol reagent).
      2. Transfer the membrane to a new container and aspirate any TBS-T residues.
      3. Pipet the ECL reagent to fully cover the membrane.
      4. Incubate for 2 min gently swirling the container, still making sure that the membrane is fully covered.
      5. Discard the ECL reagent and image your Western blot.
        Note: You can either use X-ray films (our preferred choice) or chemiluminescent imaging instruments (e.g., Chemidoc).

Notes

  1. It is of primary importance that you test the antibodies used to immunoprecipitate and/or detect each tag to exclude cross-reactivity with the other tag and also with the POI (using untagged, wild type cells). For example, while probing self-interaction between a V5-tagged and a FLAG-tagged Rad21 protein, we found that one of the FLAG antibodies did detect a band of the same size of Rad21 in untagged cells, suggesting a possible cross-reactivity with the wild type Rad21 protein (see Figure 2–figure supplement 1 in Cattoglio et al. [2019]).
  2. For a standard CoIP of endogenous proteins, you will need to identify good antibodies to immunoprecipitate and detect your POIs. If none are available, you could perform CRISPR/Cas9 endogenous tagging similar to what we do for self-CoIP, but this time tagging each of the proteins to be tested with a different tag (e.g., FLAG-Halo-tagged CTCF and V5-SNAP-tagged Rad21 in clone C59 in Hansen et al. [2019]).

Recipes

  1. Stock solutions
    1. 6 N HCl (500 ml)
      Wear eye protection and mask. Work under a fume hood. Dilute 250 ml of the concentrated acid (36.5 to 38.0%) with 250 ml of double-distilled water (ddH2O).
    2. 10x PBS, pH 7.4 (1 L)
      1. To 800 ml of ddH2O add NaCl (80 g), KCl (2 g), Na2HPO4 (14.4 g) and KH2PO4 (2.4 g). Stir to dissolve
      2. Wear eye protection and adjust the pH to 7.4 with 6 N HCl (Thermo Fisher Scientific to prepare a 6 N HCl solution dilute the concentrated acid 1:1 with ddH2O)
      3. Bring the volume up to 1 L with ddH2O and dispense 100 ml into autoclavable glass bottles
      4. Autoclave 30 min on liquid cycle
    3. 1x PBS, pH 7.4
      137 mM NaCl
      2.7 mM KCl
      10 mM Na2HPO4
      2 mM KH2PO4
      Dilute from a 10x autoclaved solution (Recipe A2) and re-autoclave
      Note: You can also purchase ready-to-use, sterile 1x PBS. Storage temperature: 15-30 °C. Shelf-life: 12 months from date of preparation.
    4. 1 M HEPES, pH 7.6 (1 L)
      1. Start with 700 ml of ddH2O. Add 238.3 g of HEPES and let dissolve
      2. Add 31.9 g of KOH (wear eye protection and mask)
      3. Bring the solution up to volume with ddH2O
      4. Filter through a bottle-top 0.2 μm filter and aliquot 40 ml into 50 ml conical tubes
      5. Store at -20 °C for up to 12 months
      Note: The 1 M solution prepared in this way has a pH of ~7.8, but a 20-fold dilution of the stock solution yields a pH of 7.6.
    5. 1 M Tris-base pH 6.8/7.5 (1 L)
      1. Start with 500 ml of ddH2O. Add 121.1 g of Tris-base and let dissolve
      2. Adjust the pH to 6.8/7.5 with 6 N HCl (wear eye protection and mask)
      3. Bring the solution up to volume with ddH2O
      4. Autoclave 30 min on liquid cycle
    6. 5 M NaCl (1 L)
      1. Start with 700 ml of ddH2O. Add 292.2 g of NaCl and stir to dissolve
      2. Bring the solution up to volume with ddH2O
      3. Autoclave 30 min on liquid cycle
    7. 2 M KCl (500 ml)
      1. Start with 300 ml of ddH2O. Add 74.6 g of KCl and stir to dissolve
      2. Bring the solution up to volume with ddH2O
      3. Filter through a bottle-top 0.2 μm filter
    8. 1 M MgCl2 (1 L)
      1. Start with 800 ml of ddH2O. Add 203.3 g of MgCl2 and stir to dissolve
      2. Bring the solution up to volume with ddH2O
      3. Autoclave 30 min on liquid cycle
    9. 1 M DTT (10 ml)
      1. Start with 8 ml of ddH2O. Add 1.54 g of DTT and invert the tube to dissolve
      2. Bring the solution up to volume with ddH2O
      3. Prepare in 1-ml aliquots and store at -20 °C for up to 12 months
    10. 10% Triton X-100 (500 ml)
      To 450 ml of ddH2O add 50 ml of Triton X-100.
    11. 0.5 M EDTA pH 8.0 (1 L)
      1. Start with 700 ml of ddH2O. Add 186.12 g of EDTA and stir (EDTA will not dissolve until the pH is near to 8.0)
      2. Adjust the pH to 8.0 with NaOH pellets (~20 g; wear eye protection and mask)
      3. Bring the solution up to volume with ddH2O
      4. Autoclave 30 min on liquid cycle
    12. 10% APS (10 ml)
      1. To 8 ml of ddH2O add 1 g of ammonium persulfate and invert the tube to dissolve
      2. Bring the solution up to volume with ddH2O
      3. Prepare in 1-ml aliquots and store at -20 °C for up to 12 months
    13. 50x cOmplete protease inhibitors stock (~ 10 ml)
      1. Dissolve 10 tablets in 10 ml of ddH2O in a 15 ml conical tube
      2. Invert the tube until completely dissolved
      3. Prepare 500-ml aliquots and store at -20 °C for up to 12 months
    14. 1 M benzamidine (10 ml; 1,000x)
      1. To 8 ml of ddH2O add 1.2 g of benzamidine and invert the tube to dissolve
      2. Bring the solution up to volume with ddH2O
      3. Prepare in 1-ml aliquots and store at -20 °C for up to 12 months
    15. 0.2 M PMSF (10 ml; 1,000x)
      1. To 9 ml of isopropanol add 348 mg of PMSF and invert the tube to dissolve
      2. Bring the solution up to volume with isopropanol
      3. Prepare in 1-ml aliquots and store at -20 °C for up to 12 months
    16. 1.23 M Bis-Tris pH 6.4/6.7 (250 ml each)
      1. To 300 ml of ddH2O add 128.7 g of Bis-Tris and stir until dissolved
      2. Bring the volume up to 400 ml and equally divide to two beakers (200 ml each)
      3. Bring the pH to either 6.4 or 6.7 with concentrated HCl (wear eye protection and mask)
      4. Bring each solution up to 250 ml with ddH2O
      5. Filter sterilize and store at room temperature for up to 12 months

  2. Buffers and other solutions
    1. Cell lysis buffer (10 ml) [final concentration]:
      100 μl of 1 M HEPES, pH 7.9 [10 mM]
      50 μl of 2 M KCl [10 mM]
      30 μl of 1 M MgCl2 [3 mM]
      1.16 gr of sucrose [~340 mM]
      1 ml of glycerol [10%]
      10 μl of 1 M DTT [1 mM]
      ddH2O to 10 ml
      1. Weight sucrose and add ~5 ml of ddH2O
      2. Add all the remaining solutions and bring volume to 10 ml with more ddH2O
      3. Filter with a syringe through a 0.2 μm filter
      4. Right prior to use, add Triton X-100 to 0.1% final (use the 10% Triton X-100 stock as a 100x concentrated solution) and preotease inhibitors (aprotinin, benzamidine, PMSF and the Roche protease inhibitor cocktail)
    2. 0.1 M CoIP buffer (500 ml) [final concentration]
      10 ml of 5 M NaCl [100 mM]
      12.5 ml of 1 M Hepes pH 7.9 [25 mM]
      500 μl of 1 M MgCl2 [1 mM]
      200 μl of 0.5 M EDTA pH 8.0 [0.2 mM]
      25 ml of 10% NP-40 alternative [0.5%]
      ddH2O to 500 ml
      To 100 ml of ddH2O add all the other solutions. Bring the volume to 500 ml and filter through a bottle-top 0.2 μm filter
    3. 0.2 M CoIP buffer (500 ml) [final concentration]
      20 ml of 5 M NaCl [200 mM]
      12.5 ml of 1 M HEPES pH 7.9 [25 mM]
      500 μl of 1 M MgCl2 [1 mM]
      200 μl of 0.5 M EDTA pH 8.0 [0.2 mM]
      25 ml of 10% NP-40 alternative [0.5%]
      ddH2O to 500 ml
      To 100 ml of ddH2O add all the other solutions. Bring the volume to 500 ml and filter through a bottle-top 0.2 μm filter
    4. 0.5% BSA in 0.2 M CoIP buffer (50 ml)
      To 50 ml of 0.2 M CoIP buffer add 250 mg of BSA. Invert the tube to mix until BSA is dissolved and filter through a 50-ml centrifuge tube top filter
    5. 20x MOPS running buffer (1 L)
      209.39 gr of MOPS
      121.1 g of Tris-Base
      20 g of SDS (wear eye protection and mask)
      7.44 g of EDTA
      1. Bring the volume to 1 liter with ddH2O and filter through a bottle-top 0.2 μm filter
      2. Store at room temperature protected from light for up to 12 months
      3. To run an SDS-PAGE, dilute to 1x with ddH2O and add sodium metabisulfute (0.475 g per 500 ml running buffer)
      4. Prepare the 1x running buffer fresh each time; do not re-use
    6. Transfer buffer (2 L)
      3.63 g of Tris-Base
      28.83 g of Glycine
      400 ml of Methanol
      1. Dissolve Tris-Base and glycine in 1 liter of ddH2O
      2. Bring the volume to 1.6 liters and add methanol
      3. Store at 4 °C for up to 12 months. We typically re-use the same transfer buffer twice before discarding it
    7. TBS-T (2 L)
      200 ml of 5 M NaCl
      10 ml of 2 M Tris pH 7.5
      2 ml of Tween-20
      1. To 1.5 liters of ddH2O add NaCl and Tris
      2. Bring the volume to 2 liters, transfer to a clean bottle, add a magnetic stirrer and Tween-20 (we typically use a P1000, dropping the whole tip filled with Tween-20 inside the bottle)
      3. Stir until all the Tween-20 is homogenously distributed and filter through a bottle-top 0.2 μm filter 
    8. 4x sample buffer (10 ml) [final concentration]
      1.6 ml of 2-mercaptoethanol [16%]
      2 ml of Tris pH 6.8 [200 mM]
      0.8 g of SDS (wear eye protection and mask) [8%]
      4 ml of Glycerol [40%]
      0.62 g of DTT [400 mM]
      0.04 gr of Bromophenol Blue [0.4%]
      1. Weight all the ingredients in a 50 ml conical tube
      2. Filter through a 0.2 μm filter 50 ml filter tube
      For a 2x sample buffer, dilute the 4x solution 1:1 with ddH2O
      For a 1x sample buffer, dilute the 2x solution 1:1 with ddH2O. Prepare 1 ml aliquots and store at -20 °C for up to 12 months
    9. Blocking solution, 10% milk (w/v) in TBS-T (100 ml)
      Dissolve 10 g of instant milk in 100 ml of TBS-T. Store at 4 °C for up to 2 weeks
    10. Blotting solution, 5% milk (w/v) in TBS-T (100 ml)
      Dissolve 5 g of instant milk in 100 ml of TBS-T. Store at 4 °C for up to 2 weeks

Acknowledgments

ASH acknowledges support from a Siebel Stem Cell Institute post-doctoral fellowship and NIH NIGMS K99 Pathway to Independence Award K99GM130896. The Tjian-Darzacq lab is supported by NIH Common Fund 4D Nucleome Program U01-EB021236 and U54-DK107980 (XD), the California Institute of Regenerative Medicine grant LA1-08013 (XD), and by the Howard Hughes Medical Institute (003061, RT). We first developed the protocol detailed here to assess the self-interaction of CTCF (Hansen et al., 2019) and of the cohesin subunit Rad21 (Cattoglio et al., 2019). We thank Jack Li for providing comments to this manuscript.

Competing interests

The authors declare no competing financial/non-financial interests.

References

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简介

[摘要 ] 蛋白质-蛋白质相互作用构成了几乎所有生物过程的分子基础。免疫共沉淀(CoIP )实验可能是探测异型和同型蛋白-蛋白相互作用的最广泛使用的方法。超分辨率显微镜的最新进展表明,几种核蛋白(例如转录因子)在空间上分布在哺乳动物细胞中的局部高浓度簇中,这表明许多核蛋白会自我相互作用。这些观察结果进一步强调了需要正交生化方法来测试是否发生自缔合,以及是否发生自缔合的机制。在这里,我们描述了一种CoIP 协议,该协议经过专门优化以测试内源标记的核蛋白(self- CoIP )的自缔合,并评估核酸在这种自相互作用中的作用。该协议已在我们手中证明了可靠和可靠,并且可以用于测试同型和异型(CoIP )蛋白质-蛋白质相互作用。

[背景 ] 蛋白质是所有生物,在那里他们服务不同的功能,包括结构,酶,信号和调节作用的基石。蛋白质通过与具有广泛亲和力的其他蛋白质,小分子(例如激素)和大分子(例如核酸,碳水化合物和脂质)相互作用来执行大多数这些任务。除了这些异型相互作用外,几种蛋白质还具有同源寡聚的能力(Paris 等,2003; Akoev 等,2004; Baisamy 等,2005; Hwang 等,2011; Chen 等,2014)。 ; Kang 等人,2017),并且自组装能力的扰动可能导致病理结果(Bourdenx 等人,2017; Yuan 等人,2018; Castle 等人,2019; Loughlin和Wilce,2019)。我们的研究主要集中在真核转录因子(TFs),启动和调节基因转录的蛋白质上。多亏了超分辨率和单分子成像技术的最新进展,我们现在才意识到,几个TF并不是均匀分布在原子核中,而是局部集中在簇中(也包括集线器或凝结物),并在它们之间快速交换(Hansen 等人,2017; Boehning 等人,2018; Cho 等人,2018; Chong 等人,2018; Dufourt 等人,2018; Mir 等人,2018){Liu,2014,Sox2的3D成像胚胎干细胞中的增强子簇; Chong,2018年,对控制基因转录的动态和选择性低复杂性域相互作用进行成像; Cho,2018年,Mediator和RNA聚合酶II簇与转录依赖的缩合物缔合; Mir,2018年,动态多因子枢纽相互作用Boehning,2018,RNA聚合酶II通过羧基末端域相分离成簇; Dufourt,2018,先驱因子Zelda通过轮毂中的瞬时相互作用对基因表达进行时间控制; Hansen,2 017,CTCF和黏附素以不同的动力学调节染色质环的稳定性} 。这些集群可以出现并通过直接的蛋白-蛋白自缔合在很大程度上维持(例如,黏附自身相互作用[ Cattoglio 等人,2019 ] )或通过的蛋白质和核酸的相互作用的组合(例如,RNA介导的CTCF自-协会[ Saldaña-Meyer 等,2014 ;Hansen 等,2019 ] )。

尽管存在一些预测蛋白质自我相互作用潜能的计算工具(Liu 等人,2013; Li 等人,2017; Zhai 等人,2017; Wang 等人,2018),但这些工具很可能对核酸视而不见酸介导的均聚反应,当然需要生化验证。体外探测蛋白质自我相互作用的可用方法包括尺寸排阻色谱,微芯片自我相互作用色谱和密度梯度离心后的生化分离,所有这些都需要纯化的重组蛋白(García 等,2003; Yusufzai 等, 2004;Saldaña-Meyer 等人,2014)。酵母或细菌双杂交系统是一种替代的体内遗传方法(Yusufzai 等,2004; Kang 等,2017),但同样,它们也无法在天然蛋白质环境中探究自我缔合。迄今为止,共免疫沉淀测定法(CoIPs )是测试相关细胞类型中发生的异型蛋白质-蛋白质相互作用的最常用方法。一些基因工程选择也可用于通过CoIP 检测同型蛋白-蛋白相互作用(图1)。一种可能性是在给定的细胞系中过表达目的蛋白(POI)的标记版本,并通过其较大的大小将其与内源性蛋白区分开(图1A)。用针对标签表位的抗体免疫沉淀后,使用识别标签蛋白和野生型蛋白的抗体进行蛋白质印迹。免疫沉淀样品中野生型条带的出现被认为是自我相互作用的证据(Pant 等,2004;He 等,2008; Kang 等,2017)。这种方法有几个缺点:它使用一种外源表达的蛋白质,可以改变其生理行为,并且不能排除观察到的野生型条带仅仅是标记蛋白质的降解产物。为了解决后者,一种更可靠的方法可能是转染两个构建体,每个构建体表达标记有不同表位的POI(图1B中的“ TAG1”和“ TAG2”)。在这种情况下,如果TAG1标记的蛋白也拉下TAG2蛋白,则可以得出结论POI确实是自相互作用的。可替换地,在混合的体内和IN- 在体外方法中,将过表达用一个表位标记的POI的细胞裂解物与用另一表位标记的重组POI一起孵育(Saldaña-Meyer 等,2014)。该方法的优点是可以轻松筛选POI的截短形式以识别负责自我缔合的区域,但又依赖于过表达并且需要纯化的蛋白质。替代CoIP 的基于成像的方法来评估外源表达蛋白的自身相互作用是双分子荧光互补测定法,其中POI与分裂的荧光蛋白(例如GFP或YFP)的一个或另一半融合( Miller 等,2015)。仅当蛋白质自缔合时,荧光蛋白质才会重构并发出荧光(Kang 等人,2017)。该测定不能克服过表达的问题,并且还受到荧光重建反应不可逆性的限制,荧光不可逆反应会引入偏差并使该方法对自相互作用的动力学视而不见。



C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1902918--1289 Claudia Cattoglio 809941 \ Figs jpg \ Figure1.jpg

图1.通过CoIP 测试蛋白质自相互作用。通过共免疫沉淀技术对细胞进行基因工程和测试POI的自缔合的不同策略。A. 单个质粒编码标记的POI是TRAN 小号染到除了来自内源性启动子(1)表示的野生型蛋白的细胞。如果POI自相互作用,则野生型POI将与其自身(未显示)以及与外源表达的标记版本(2)相关联。甲免疫共沉淀,使用针对该标签也将下拉野生型POI(3)的抗体的实验。如果标签足够大,则可以使用抗POI的抗体在Western blot实验中通过大小将标记的POI与野生型区分开(4)。B. 与(A)中相同,但是这次转染了两个单独的质粒,每个质粒编码带有不同表位(TAG1和TAG2)的POI(1)。将免疫沉淀的材料进行SDS-PAGE,然后进行蛋白质印迹,并使用针对每个标签的抗体评估自身相互作用(4)。C. 我们用2种不同的表位标记内源POI的策略。将CRISPR / Cas9靶向构建体(用于表达Cas9和选择的gRNA)与两个供体质粒一起转染,每个供体质粒均包含选择的标签(TAG1或TAG2),其侧翼为与内源POI同源的基因组序列(LHR,左同源区和RHR,右同源区)(1)。选择并表征含有每个内源性等位基因的克隆,这些等位基因标记有两个表位(1 ' )之一。如果POI自相互作用(2),则使用针对TAG1的抗体进行CoIP 实验也会拉低TAG2-POI(3)。对免疫沉淀的材料进行SDS-PAGE,然后进行蛋白质印迹,并使用针对每个标签的抗体评估自身相互作用(4)。可以将对RNA(RNase),DNA(DNase)或二者(苯甲酸酯酶)特异的核酸酶包括在实验中,以测试自结合是否依赖于蛋白质-蛋白质相互作用和/或由核酸介导。IN,输入裂解物;TAG IP,免疫沉淀材料



我们测试以生理浓度表达的TF或​​其他核蛋白的自缔合的方法是通过CRISPR / Cas9编辑目标细胞系的基因组(参考文献1; Roberts 等人,2017; Aird 等人,2018; Haupt 等,2018; Sharma 等,2018; Tasan 等,2018; Zhang 等,2018; Liu 等,2019),并在每个编码TF的等位基因中引入了两个不同的标签(通常为FLAG- Halo和V5-SNAP f )(Cattoglio 等,2019; Hansen 等,2019)(图1C)。可以通过与CRISPR / Cas9并行提供2个单独的同源性修复构建体来尝试双重标记,但通过使用来自第一个标记的杂合克隆并重新靶向,我们通常在更高的成功率下在一个单独的实验中依次标记每个等位基因。 “野生型”等位基因(实际上,这通常是伪wt 等位基因,其中Cas9在切割位点引入了插入缺失;可以利用它来设计另一套专门针对未标记等位基因的指导RNA)。然后,使用双标签等位基因,我们可以通过免疫共沉淀(self- CoIP )来探查TF是否自我相互作用,并进一步研究这种自我缔合是否是通过蛋白质-蛋白质相互作用和/或核酸进行介导的。或没有特定的核酸酶。我们在这里提供了执行自我CoIP的详细说明,同时您可以参考我们以前的出版物以了解内源性双重标记策略(Cattoglio 等,2019; Hansen 等,2019)。该方法无法区分直接和间接自我相互作用,并且由于它需要细胞裂解和较长的孵育时间,因此无法准确估计活细胞中发生的自我缔合的数量,而是给出了较低的界限。尽管如此,内源性标记避免了源自过度表达的假象(Hansen 等人,2017; Shao 等人,2018),并且,如果所有部署的抗体都是特异性的(见注1 ),则此处详述的self-CoIP 方法仅需标准实验室设备和试剂,并且它与数个TF一起在我们手中一直稳定且可重复地工作。尽管此协议可靠地检测了核蛋白的自缔合,但我们还是常规地使用它来在标准CoIP 环境中在细胞核和细胞质中测试异型蛋白-蛋白相互作用(请参见注释2)。

关键字:免疫沉淀反应, 免疫沉淀, 免疫共沉淀, 免疫共沉淀, 自身相互作用, Cas介导内源性标记

材料和试剂


 


移液器技巧
150毫米TC处理过的培养皿(Corning TM 430599,Thermo Fisher Scientific,目录号:08-772-24)
细胞刮板(Thermo Fisher Scientific,Fisherbrand TM ,目录号:08-100-241)
Cuvettes (Thermo Fisher Scientific,Fisherbrand TM ,目录号:14-955-127)
MagneSphere ® 技术12位置,1.5毫升磁选支架(Promega,目录号:Z5342)
最小的空凝胶盒,1.0毫米(Thermo Fisher Scientific公司,Invitrogen TM ,目录号:NC2010)
空凝胶盒式梳子,迷你,1.0 mm,10孔(Thermo Fisher Scientific,Invitrogen TM ,目录号:NC3010)
安玛西亚TM PROTRAN ® Western印迹膜(Millipore公司Sigma,目录号:GE10600041),保存于室温下。保质期:由制造商指定)
3 MM防污纸(Whatman,目录号3030-917)
Falcon TM 圆底聚丙烯5 ml管(Corning TM 352063,Thermo Fisher Scientific ,目录号:14-959-11A)
50 - 毫升锥形管(康宁TM 430291,赛默飞世尔科技,产品目录号:05-538-55A)
刀片(VWR ® ,目录号:55411-050)
Steriflip -GP无菌离心管顶部过滤器(M illipore Sigma,目录号:SCGP00525 )
10毫升注射器,Luer-Lock TM (Becton Dickinson,目录号:302995)
22G PrecisionGlide TM 针(Becton Dickinson,目录号:305156)
1.5 ml低保留微量离心管(Phenix ,目录号:MH-815S)
12×24毫米的圆底玻璃管(Covaris ® ,目录号:520056)
用0.22一次性无菌瓶顶部过滤器μ 米膜(康宁TM 430513-500 ml或430626- 150毫升,赛默飞世尔科技,产品目录号:09-761-52或09-761-56)
CL- XPosure 胶片(Thermo Fisher Scientific,目录号:34091 )
用于说明性实验的抗体如下:
中号乌斯正常的IgG(Abcam公司,目录号:ab37355)
中号乌斯α FLAG(Millipore公司Sigma公司,目录号:F3165)
HEPES(赛默飞世尔科技,费舍尔BioReagents公司,目录号:BP310500),小号撕在室温下。小号HELF寿命:由制造商指定
盐酸(HCl,Thermo Fisher Scientific,目录号:A144-500)
氯化钾(KCl的,赛默飞世尔科技,Fisher化学公司,目录号:P217-10),小号撕在室温下。小号HELF寿命:由制造商指定
氢氧化钾(KOH,赛默飞世尔科技,Fisher化学公司,目录号:P250-500),小号撕在室温下。小号HELF寿命:由制造商指定
氢氧化钠颗粒(氢氧化钠,赛默飞世尔科技,Fisher化学公司,目录号:S318-100),小号撕在室温。保质期:由制造商指定
六水合氯化镁(MgCl 2· 6H 2 O,赛默飞世尔科技,Fisher化学公司,目录号:M33-500),小号撕在室温。保质期:由制造商指定
氯化钠(NaCl,赛默飞世尔科技,产品目录号:S271-10),小号撕在室温。保质期:由制造商指定
将2-丙醇(赛默飞世尔科技,产品目录号:A451-4),小号撕在室温。保质期:由制造商指定
d-蔗糖(C 12 H ^ 22 ö 11 ,赛默飞世尔科技,费舍尔BioReagents公司,目录号:BP220-212),小号撕在室温。保质期:由制造商指定
甘油(C 3 H ^ 8 ø 3 ,赛默飞世尔科技,Fisher化学公司,目录号:G33-20),小号撕在室温。保质期:由制造商指定
DTT(C 4 ħ 10 ö 2 s ^ 2 ,GOLDBIO,目录号:DTT10),š torage温度:-20℃。保质期:由制造商指定
完整TM ,无EDTA的蛋白酶抑制剂混合物(西格玛Millipore公司,罗氏,目录号:11873580001),š torage温度:4℃(片剂),-20℃(溶解,见食谱)
的Triton TM X-100(Millipore公司Sigma,目录号:T9284),小号撕在室温。保质期:由制造商指定
抑肽酶(Millipore公司Sigma,目录号:A6279),š torage温度:4℃。保质期:由制造商指定
20毫克/毫升牛血清白蛋白(BSA)(New England Biolabs公司,目录号:B9000S) ,š torage温度:-20℃。保质期:由制造商指定。用于创建用于蛋白质定量的BSA标准
BSA(Millipore公司Sigma,目录号:A7906),š torage温度:4℃。保质期:由制造商指定。用于预清除磁珠。
苯甲脒(Millipore公司Sigma,目录号:B6506),š torage温度:4℃(粉),- 20℃(溶液,参见食谱)。保质期:由制造商指定
PMSF(Millipore公司Sigma公司,罗氏,目录号:11359061001),š torage温度:室温。保质期:由制造商指定
EDTA(C 10 ħ 18 Ñ 2 的Na 2 ö 10 ,赛默飞世尔科技,Fisher化学公司,目录号:S311-500),š torage温度:室温。保质期:由制造商指定
NP-40的替代,蛋白质级洗涤剂,10%溶液(Millipore公司Sigma,目录号:492018),š torage温度:4℃。保质期:由制造商指定
的Benzonase ®核酸酶HC(Millipore公司西格玛,Novagen公司,目录号:71205-3),š torage温度:-20℃。保质期:由制造商指定
Bio-Rad公司的蛋白质测定染料试剂(Bio-Rad公司,目录号:5000006 ),š torage温度:4℃。保质期:由制造商指定
的Dynabeads TM 蛋白A或G(赛默飞世尔科技,Invitrogen公司TM ,目录号:10008D或10009D ),š torage温度:4℃。保质期:自生产之日起24个月
Precision Plus Protein TM 所有蓝色预染色蛋白标准品(B io- R ad ,目录号:1610393)
MOPS(C 7 ħ 15 NO 4 S,赛默飞世尔科技,费舍尔BioReagents公司TM ,目录号:BP308-500 ),小号撕在室温。保质期:由制造商指定
Tris碱(C 4 ħ 11 NO 3 ,赛默飞世尔科技,费舍尔BioReagents公司TM ,目录号:BP152-5 ),小号撕在室温。保质期:由制造商指定
SDS(Bio-Rad公司,目录号:1610301 ),小号撕在室温。保质期:由制造商指定
乙醇(Decon L abs,Koptec 纯乙醇– 190 Proof,目录号V1101)
乙是-T RIS (C 8 ħ 19 NO 5 ,Millipore公司Sigma,目录号:B9757),小号撕在室温。保质期:由制造商指定
焦亚硫酸钠(Na 2 S 2 O 5 ,密理博西格玛(Millipore Sigma),目录号:S9000),在室温下储存。保质期:由制造商指定
TEMED(Bio-Rad公司,目录号:1610800 ),š torage温度:4℃。保质期:由制造商指定
过硫酸铵(APS)(Bio-Rad公司,目录号:1610700 ),š torage温度:室温(粉),-20℃(溶液,见ř ecipes)。保质期:由制造商指定(粉末),6个月或更长时间(溶液)
ProtoGel (30%)37.5:1至丙烯酰胺双丙烯酰胺溶液(国家诊断,目录号:EC-890),š torage温度:4℃。保质期:由制造商指定
超纯TM 水(赛默飞世尔科技,费舍尔BioReagents公司TM ,目录号:10977023 ),小号撕在室温。保质期:由制造商指定
甘氨酸(C 2 H ^ 5 NO 2 ,赛默飞世尔科技,费舍尔BioReagents公司TM ,目录号:BP381-5 ),小号撕在室温。保质期:由制造商指定
甲醇(CH 4 O,赛默飞世尔科技,Fisher化学公司,目录号:A452-4 ),小号撕在室温。保质期:由制造商指定
吐温® -20(M illipore西格玛,Calbiochem公司,产品目录号:655205),小号撕在室温。保质期:由制造商指定
即时脱脂牛奶,巴氏杀菌特级(签字厨房),小号撕毁在室温下。保质期:由制造商指定
2-巯基乙醇(M illipore Sigma,目录号:M3148),小号撕在室温。保质期:由制造商指定
溴酚蓝钠盐(C 19 H 9 Br 4 NaO 5 S,Milli Sigma,目录号:B5525),在室温下保存。保质期:由制造商指定
磷酸二氢钾(Na 2 HPO 4 ,Thermo Fisher Scientific,Fisher Chemical,目录号:P285-500)
无水磷酸氢二钠(KH 2 PO 4 ,Thermo Fisher Scientific,Fisher Chemical,目录号:S375-212)
西方闪电PLUS-ECL(珀金埃尔默,目录号:NEL103E001EA) ,š torage温度:4℃。保质期:由制造商指定
70%乙醇
6 N HCl(请参阅食谱)
10x PBS(请参阅食谱)
1x PBS,pH 7.4(请参阅食谱)
1 M HEPES(请参阅食谱)
1 M Tris-base pH 6.8 / 7.5(请参阅食谱)
5 M NaCl(请参阅食谱)
2 M KCl (请参阅食谱)
1 M MgCl 2 (请参阅食谱)
1 M DTT(请参阅食谱)
10 %的Triton X-100(请参阅食谱)
0.5 M EDTA pH 8.0(请参见配方)
10%APS(请参阅食谱)
50x cOmplete 蛋白酶抑制剂库存(请参阅食谱)
1 M苯甲idine(请参阅食谱)
0.2 M PMSF(请参阅配方)
1.23 MB 为-T ris pH 6.4 / 6.7(请参阅配方)
细胞裂解缓冲液(请参阅食谱)
0.1 M CoIP 缓冲区(请参阅食谱)
0.2 M CoIP 缓冲区(请参阅食谱)
0.5 M的BSA在0.2 M CoIP 缓冲液中(请参阅配方)
20x MOPS运行缓冲区(请参阅食谱)
传输缓冲区(请参见食谱)
TBS-T(请参阅食谱)
4x样品缓冲液(请参见配方)
阻止解决方案(请参阅食谱)
印迹解决方案(请参阅食谱)
 


设备


 


-80°C冷冻室
移液器(P20,P200,P1000)
DynaMag TM -15磁铁(Thermo Fisher Scientific,目录号:12301D)
Focused- 超声发生器(Covaris ® ,型号:S220)与12 X 24管适配器(Covaris ® ,目录号:500199)
注意:您可以使用任何类型的超声波仪(例如,超声波处理器)来替换本仪器。我们建议在没有核酸酶消化的情况下进行超声处理以溶解染色质。你与胞质蛋白(注上工作时可以跳过超声步骤小号,或者用核酸整个样本消化TEP C5)。


冷藏台式离心机(Eppendorf,型号:5810R)
冷藏微量离心机(Eppendorf,型号:5418R )
紫外可见分光光度计(岛津制作所,型号:UV-1900)
Fisherbrand TM Isotemp TM 数字块式加热器(Thermo Fisher Scientific,目录号:88-860-021)
XCell SureLock TM 微型电池(Thermo Fisher Scientific,目录号:EI0001)
电泳电源(Thermo Fisher Scientific,型号:EC-105);用于SDS-PAGE
迷你反式印迹® 电泳转移细胞(B IO- ř 广告,目录号:1703930)
PowePac TM 基本电源(Bio-Rad ,目录号:1645050);用于转移
粘土亚当斯章鱼混合器(Becton Dickinson); 用于裂解物
Rocker平台(Bellco Biotechnology,型号:7740-10010):用于用抗体过夜印迹蛋白质印迹膜
台式轨道振动器(Thermo Fisher Scientific,型号:MaxQ TM 2000)
 


程序


 


收集细胞
使您感兴趣的细胞系达到80%融合。
注意:对于小鼠胚胎干细胞,我们通常从两个150 mm融合的组织培养板(约30 x 10 6 -40 x 10 6 总细胞)开始。使用的培养板数量将取决于您可以从每个培养板上获得多少个细胞(例如,小鼠胚胎干细胞在3D菌落中生长,因此产量非常高)以及要进行免疫沉淀的蛋白质的细胞丰度。


对于贴壁细胞:
吸出媒体。
用冰冷的1x PBS洗涤两次:
将足够的PBS倒在板上以覆盖它并旋转,以除去培养基残留物。
吸出PBS或将板倒置以丢弃它。
将板转移到冰上。
注意:从现在开始,除非另有说明,否则始终将样品和缓冲液置于冰中。


用冰冷的1x PBS(添加PMSF,苯甲idine和抑肽酶)刮擦细胞(使用蛋白酶抑制剂的原液作为1,000x)。通常,每150毫米平板使用3-4毫升PBS。
将刮取的细胞转移到15 ml锥形管中(如果最终体积超过12 ml,则转移50 ml)。
通过用另外的3-4 ml 含蛋白酶抑制剂的1 x PBS 洗涤刮板来收集所有剩余的细胞,然后转移到相同的15 ml锥形管中。
在4°C和300 xg 的冷藏台式离心机中沉淀细胞10分钟。
吸出上清液并在液氮中速冻细胞沉淀,并储存在-80°C直至准备进行。
对于悬浮生长的细胞:
将细胞从培养容器转移到50 ml锥形管中;用1x PBS洗涤培养皿一次,以收集所有剩余的细胞。
在25°C和300 xg 的台式离心机中沉淀细胞5分钟。
将细胞沉淀转移到冰上。
用40毫升冰冷的1x PBS洗涤一次:
将沉淀重悬于PBS中。
在4°C和300 xg 的冷藏台式离心机中沉淀10分钟。
将沉淀重悬于20 ml冰冷的1x PBS中,其中加入PMSF,苯甲idine和抑肽酶。
在4°C和300 xg 的冷藏台式离心机中沉淀细胞10分钟。
吸出上清液并在液氮中速冻细胞沉淀,并储存在-80°C直至准备进行。
 


打开Covaris 超声仪并设置为4°C
注意:超声仪需要约30-45分钟才能完成脱气和冷却。


 


裂解核(所有体积均指2个150毫米平板或30-40百万个细胞)
制备细胞裂解缓冲液(见ř ecipe B1; 700 μ 升对于2x 150毫米的板,或30-40百万个细胞),加入蛋白酶抑制剂(PMSF,抑酶肽,苯甲脒,一个d罗氏完整抑制剂-50x股票)和将Triton X-100的最终浓度调节为0.1%(使用10%的原液作为100x)。
重悬700每个粒料μ 升的新鲜制备的细胞裂解缓冲液使用P1000吸管。
将裂解液转移至低保留度的1.5 ml管中,并在4°C下摇动8分钟以裂解(例如,在冷室中使用旋转器)。
在4°C和3,000 xg 的冷冻微量离心机中离心3分钟。
可选:将上清液保留为“细胞质级分”。
注意:此协议专门针对核蛋白进行了优化,但是您也可以在细胞质级分上执行self-CoIP 。


首先用P1000除去上清液,然后用P200除去上清液,而不会干扰核沉淀。
制备0.1M的免疫共沉淀缓冲液(见ř ecipe B2; 700 μ 升对于2x 150毫米的板,或30-40百万个细胞),加入蛋白酶抑制剂(PMSF,抑酶肽,苯甲脒,和罗氏完全inhibitors- 50 X 股票) 。
重悬700中的每个核沉淀μ 升的0.1M的免疫共沉淀缓冲液中。
注意:如果您不希望进行核酸酶消化,请直接将核沉淀重悬于0.2 M CoIP 缓冲液中,超声处理裂解物,然后从步骤s C10 跳至C17。


将重悬的细胞核转移到12 x 24 mm Covaris 管中。
对每个样品进行超声处理(峰值功率:100 W,周期/突发:200,占空比:20,处理20秒的8次重复,然后延迟40秒)。
测量样品的体积,同时将其转移回1.5毫升低保留管中。
用0.1 M CoIP 缓冲液加蛋白酶抑制剂将体积调至2 ml 。
将样品等分,以两个管,1毫升被留下未处理的和1ml与治疗乙enzonase (1 μ 升每ml裂解物)。
注意:Benzonase会同时消化DNA和RNA,因此会告诉您自我相互作用是否主要是由蛋白质-蛋白质相互作用介导的,还是需要核酸。如果自缔合是依赖于核酸,可以测试是否RNA或DNA都会参与其中,取代的Benzonase 与任一RNaseI 或DNA酶I (你将需要调整用于消化缓冲液DNA酶I )。
岩石在4℃下进行3至4小时,以实现完全的Benzonase 消化。
在4°C的冷冻微量离心机中短暂旋转,以拉下液体。
添加5M的NaCl以使最终盐浓度至0.2M(即,20.4 微升,并立即反转管每1ml裂解物)。
在4°C摇动30分钟,以实现完全核裂解。
在4°C下以最大速度旋转冷藏微量离心机20分钟。
将上清液转移至新的低保留1.5 ml管中,并通过Bradford分析进行定量。
 


定量核裂解液
打开紫外/可见分光光度计。
准备足够的Bradford试剂来量化所有的溶胞产物加6倍额外的样品以制备BSA标准曲线(空白,2.5 微克,5 微克,7.5 微克,12 微克)。每个样品将需要1 ml的Bradford(用ddH 2 O 稀释Bio-Rad蛋白测定染料试剂1:5 )。
准备BSA标准品,将47.5 微升的0.2M的免疫共沉淀缓冲到2.5 微升的20毫克/毫升BSA(NEB)。这会给你一个1 微克/ 微升BSA解决方案。
将Bradford试剂分装到1.5 ml试管中,如下所示:
1毫升(空白)


997.5 微升布拉德福德+ 2.5 微升BSA(2.5微克标准)


995 微升Bradford + 5 微升BSA(5微克标准液)


992.5 微升布拉德福德+ 7.5 微升BSA(7.5微克标准)


988 微升Bradford + 12 微升BSA(12微克标准品)


998 微升布拉德福德+ 2 微升样品#1 /未处理


998 微升布拉德福德+ 2 微升样品#1 / 的Benzonase


对于需要定量的样本数量。


加入BSA /裂解液后,立即颠倒每个试管进行混合。
将每个试管的内容物倒入塑料比色杯中,读取595 nm处的吸光度(用空白溶液自动将仪器清零)。
使用提供的模板 补充表S1用BSA读数建立标准曲线,并内插样品浓度。
 


设置您的自我C oIP 实验并预先清除裂解物和珠子
我们在这里说明了一个典型的自我CoIP 实验,以探索黏着蛋白亚基Rad21 的自我关联及其对核酸的依赖性。具体来说,我们正在使用来自小鼠胚胎干细胞的核裂解物,其具有内源性双重标记的Rad21蛋白(最初在Cattoglio 等人(2019)中描述的B4克隆。该细胞系包含Rad21-SNAP f -3xFLAG等位基因和Rad21- Halo-V5等位基因(图2A):我们将用FLAG抗体下拉从第一个等位基因表达的蛋白质(图2B),并检查在Western blot中是否也使用V5抗体免疫沉淀了第二个等位基因产生的V5标记蛋白b 很多(图2C),无论在未处理的裂解物,并与处理过的溶胞产物的Benzonase 消化核酸。我们也将检查实验印迹针对免疫沉淀的FLAG蛋白质(图2D)的总IP效率。


 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1902918--1289 Claudia Cattoglio 809941 \ Figs jpg \ Figure2.jpg


图2. Rad21 的自我CoIP Rad21自我CoIP 分析的实验工作流程和结果在这里用于说明协议。C和D中的数据是根据Cattoglio 等人的原始图2F修改的。(2019)。有关详细说明,请参见过程E。IN,输入核裂解物;UT,未处理的裂解物;苯:苯甲酸酶处理的裂解物。


 


为您的实验创建一个方案,如下所示:
样品号(需要毫克)             


样品1:mESC / 未经处理的输入(0.2 mg )                                                                                   


样品2:mESC / 未经处理的IgG (1 mg )


样品3:mESC /未经处理的α 标记(1 mg)             


样品4:mESC / 苯甲酸酯酶输入(0.2 mg)


样品5:mESC / 苯甲酰酶_IgG (1 mg)


样品6:mESC的/ benzonase核酸_ α FLAG(1毫克)


在这种情况下,每种情况(未经处理和经苯甲酸酶处理)将需要2.2 mg核裂解物。


注:正常IgG的样品(来自免疫前血清中纯化免疫球蛋白)控制非特异性下拉和它必须是你的相同物种的α FLAG抗体(例如,小鼠抗FLAG抗体和正常小鼠IgG)。


用添加蛋白酶抑制剂(PMSF,抑肽酶,苯甲idine 和Roche cOmplete 抑制剂)的0.2 M CoIP 缓冲液将实验所需的裂解液稀释至1 mg / ml 。如果体积超过1.5毫升,请将稀释的裂解物移至圆底试管中(图3a)。
在我们的说明性实验中:


条件浓度(表S1)对于2.2 mg 0.2 M CoIP 缓冲液至1 mg / ml                                         


mESC的/未处理的2.5毫克/毫升880 μ 升1320 μ 升                                         


mESC的/ benzonase核酸2.8毫克/毫升786 μ 升1414 μ 升                                         


 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1902918--1289 Claudia Cattoglio 809941 \ Figs jpg \ Figure3.jpg


图3.分步自CoIP 协议。该图说明了免疫沉淀目标蛋白所需的所有步骤,在本文中进行了详细描述。


 


带珠子的Preclear裂解物可减少非特异性结合(请注意,这些珠子将在预分离结束时丢弃)(图3b)。
洗净蛋白A或G D ynabeads 。
注意:根据一抗的种类和IgG亚型选择蛋白A或G(例如,将G蛋白用于兔和小鼠IgG2a抗体,将A蛋白用于豚鼠)。检查磁珠制造商的规格表,以确认您的选择。


大力重悬D ynabeads,直到均匀分散。
对于各条件,吸管20 微升的d ynabeads 每裂解物的毫克(例如,44 微升2.2毫克)到低保持1.5ml试管中。
重悬在500 微升的0.2M的免疫共沉淀缓冲液中。
插入在磁铁分离架上的管,并等待对于5 分钟的溶液来清除。
用移液管吸出CoIP 缓冲液,从磁铁上移开试管,然后再次洗涤(步骤E3a -iii 至E3a -v )。
从磁铁上移开试管,然后使用稀释的裂解液重悬磁珠,并将其与其余核蛋白一起转移到圆底试管中。
在4°C下用小珠摇动裂解液至少2 h。
使用BSA的Preclear磁珠(请注意,您将使用这些磁珠执行实际的下拉实验)(图3e-3f)
洗净蛋白A或G D ynabeads ; 您将准备一个单独的D ynabeads 池,以用于所有样本。
大力重悬D ynabeads,直到均匀分散。
在1.5毫升管,吸管20 微升的d ynabeads 每个样本(例如,20 微升为6个样品:120 微升)。
注:Ÿ 欧不会吸管d ynabeads 您的输入样本,但我们包括输入预清除珠时以后,以容纳移液误差。


重悬于1 ml的0.2 M CoIP 缓冲液中。
插入管在1.5毫升磁选支架,并等待对于5 分钟的溶液来清除。
用移液管吸出CoIP 缓冲液,从磁铁上移开试管,然后再次洗涤(步骤E4a -iii 至E4a -v )。
从磁铁上取下试管,然后将磁珠重悬在含0.5%BSA的0.2 M CoIP 缓冲液中(请参阅配方B4)。使用缓冲当量的量的你的珠10倍的体积(例如,1 ,200 微升120 BSA缓冲液微升的小珠)。
在4°C下用BSA摇珠过夜。
注意:为方便起见,我们在将裂解物与抗体一起孵育的同时,将过夜用BSA的珠子进行了预清洁,但是几个小时也足够。


 


将抗体添加到预先清除的裂解物中(图3c-3d)。
在4°C 下短暂旋转预澄清的裂解液,以除去管盖上的液滴。
取下卡口盖,然后将管子插入DynaMag TM 磁力架。
等待为5分钟,使该溶液澄清。             
注意:在进行此过程以及随后的所有磁分离过程中,我们希望将磁架转移到冰箱中,等待溶液清除,以始终保持样品冷却。


将上清液转移到新的试管中而不破坏珠子。
根据IP需要准备尽可能多的1.5 ml低保留管(例如,对于我们的6个样品)。
将预澄清的裂解液分配到新试管中。在我们的说明性实验中,这将对应于:
样品2-3和5-6:移液管1 ml(相当于1 mg)


样品1和4(输入):100-150吸管微升(相当于0.1-1.15毫克)


添加特异性抗体(4 μ 克由混合每裂解物的毫克),并立即转化管。在我们的说明性实验中:
样品1(mESC / 未经处理的输入):无抗体                                                                                   


样品2(mESC的/ untreated_IgG ):4 μ 克小鼠正常的IgG的


样品3(mESC的/ untreated_ α FLAG):4 μ 克小鼠的α FLAG


样品4(mESC / benzonase_input ):无抗体


样品5(mESC的/ benzonase_IgG ):4 μ 克小鼠正常的IgG的


样品6(mESC的/ benzonase核酸_ α FLAG):4 μ 克小鼠的α FLAG


在4°C下用抗体旋转/摇动裂解物过夜。
注意:孵育抗体过夜时,我们可获得最佳结果,但是您可以测试抗体并尝试缩短时间。


 


将抗体与预先清除的磁珠结合(图3g-3i)。
取回样品和经过BSA预处理的珠子,并在4°C 下短暂旋转,以除去管盖上的液滴。
将带有预清除磁珠的试管放入磁力分离器架中。
等待为5分钟,使该溶液澄清。             
从在0.2M的磁体,重悬珠子去除管免疫共沉淀用缓冲液抑制剂(50 μ 升每样本;在我们的实验说明,50 微升×6个样品= 300 μ 升总)。
Distibute 50 μ 升的悬浮珠粒到每个样品被免疫沉淀(输入除外),并立即倒置以混合。
用抗体和珠子在4°C 旋转/摇动裂解物最少2 h。
将输入样品放在冰上。
 


洗涤免疫沉淀的样品(IP样品)以去除非特异性结合(图3j)。
准备足够的冰冷的0.2M 免疫共沉淀缓冲液加蛋白酶抑制剂至8倍与400洗每个IP样品微升缓冲液(例如,4个IP样本x 400 微升×8 =12.8毫升0.2 M的免疫共沉淀缓冲液;输入除外)。
在4°C 短暂旋转IP样品,以除去管盖上的液滴。
将IP样品的试管插入1.5 ml的磁力架中。
等待为5分钟,使该溶液澄清。此时,您的免疫沉淀蛋白会通过抗体附着在磁珠上。             
使用P1000 移液器卸下CoIP 缓冲液,而不会干扰珠子。
从磁铁上取下试管,然后放回冰上。
加入400μl 的0.2 M CoIP 缓冲液。
注意:确保珠子完全重悬。我们通常以低速涡旋。


重复步骤H4-H7进行总共8次洗涤。
最后一次洗涤后,还用P20 清除所有残留的缓冲液。
 


洗脱免疫沉淀的物质并准备用于SDS-PAGE的输入和样品(图3k-3l)。
从磁铁上取下试管,然后放回冰上。
洗脱免疫沉淀从珠中加入20蛋白μ 升1X SDS上样缓冲液(配方B8)的。
注意:洗脱量取决于您有多少个样品。我们在这里说明使用10孔丙烯酰胺凝胶的实验,该凝胶可以轻松容纳17μl 。我们还使用12孔或15孔凝胶,并相应地调整洗脱体积或装载较少的材料。


涡旋重悬。
在设定为98 °C 的加热块中将小珠煮沸5分钟。
短暂旋转。
将试管放入1.5 ml的磁力架中。
等待为5分钟。您洗脱的蛋白质现在在上清液中。
将上清转移到新的低保留1.5在冰上ml管(体积要稍多于20 微升)。您将在一块凝胶上上样此未稀释样品的量为17μl ,以估计自CoIP 效率(图2C)。
准备一个新的组管18 μ 升1的X SDS上样缓冲液,并添加2 μ 升洗脱的材料制成。你将加载17 微升此的上的第二凝胶1:10稀释来估计IP效率(图2D)。
准备0.75%的输入以与IP样本一起加载。
混合22 μ 升输入裂解物与25 μ 升2×SDS上样缓冲液和3 μ 升0.2M的免疫共沉淀缓冲液。
注:这削弱了你的inpute 裂解物0.44 μ g ^ / μ 升。装载17 μ 升该稀释的会给你〜7.5 μ 克,其对应于起始原料(1毫克)0.75%。


在设定为98 °C 的加热块中煮5分钟。
短暂旋转,然后将试管放在冰上。
 


通过SDS-PAGE和Western印迹评估自我CoIP 和CoIP 效率
P repare SDS-PAGE丙烯酰胺凝胶
注意:如果更方便,则可以从Invitrogen 购买预制的NuPAGE Bis-Tris凝胶。


根据POI的大小选择凝胶百分比。凝胶迁移图可从ThermoFisher Scientific网站上获得(我们的凝胶配方的行为类似于Invitrogen TM 的NuPAGE Bis-Tris预制凝胶)(参考文献33 )。对于Rad21,我们使用9%的凝胶。
检查表1的食谱。
 






表1.使用此表准备您的SDS-PAGE凝胶(所有指定的体积均用于1毫米暗盒,1凝胶)


分离胶


 


 


 


 


 


试剂


12%


10%


9.5%


9%


8%


1.23 M Bis-Tris pH 6.7


1.8毫升


1.8毫升


1.8毫升


1.8毫升


1.8毫升


30%丙烯酰胺


2.52毫升


2.1毫升


2.0毫升


1.89毫升


1.68毫升


二次蒸馏H 2 O


1.93毫升


2.35毫升


2.45毫升


2.56毫升


2.77毫升


10%APS


42微升


42微升


42微升


42微升


42微升


特美


12微升


12微升


12微升


12微升


12微升


总容积:


6.3毫升


6.3毫升


6.3毫升


6.3毫升


6.3毫升


堆积胶


 


 


 


 


 


试剂


4%


 


 


 


 


1.23 M Bis-Tris pH 6.4


710微升


 


 


 


 


30%丙烯酰胺


330微升


 


 


 


 


二次蒸馏H 2 O


1.43毫升


 


 


 


 


10%APS


20微升


 


 


 


 


特美


7微升


 


 


 


 


总容积:


2.5毫升


 


 


 


 


 


将两个1毫米凝胶盒放置在稳定的直立位置。
在15毫升的锥形管中,开始准备分离凝胶,并按照表1中指定的顺序添加试剂(Bis-Tris,丙烯酰胺和水)。
上下充分混匀,最后加入APS和TEMED。
及时混合并吸取到凝胶盒中,为堆叠凝胶留出足够的空间。
立即用喷水瓶在凝胶上覆盖70%乙醇。这将使您的凝胶正面整齐而笔直。
等待聚合完成。
注意:固化后,凝胶前沿将显示为整齐的线条,可与上面分层的乙醇区分开来。


抽吸乙醇,并使用喷水瓶用ddH 2 O 彻底洗涤凝胶。
吸出过量的DDH 2 O.
在一个新的15 ml锥形管中,开始准备堆积凝胶,并按照表1中指定的顺序添加试剂(Bis-Tris,丙烯酰胺和水)。
上下充分混匀,最后加入APS和TEMED。
及时混合并吸取到分离凝胶顶部的凝胶盒中,一直到盒顶部。
缓慢放置选定的梳子(在本例中为10孔梳子),确保在孔下面没有气泡形成。
注意:我们通常在插入梳子时用P1000吸去多余的堆积凝胶,然后将其转移回我们准备的15 ml试管中,以免丙烯酰胺溅到工作台上。


等待聚合完成。
运行SDS-PAGE
将凝胶转移到正在运行的盒中(每个盒可容纳2个凝胶)并将其锁定到位。
注意:切记剥离凝胶盒底部的白色胶粘剂。


根据配方B5稀释20倍MOPS运行缓冲液。
注意:切记要添加焦亚硫酸钠。


从凝胶盒中取出梳子。
将运行缓冲液一直充满到两个纸盒之间的内部腔室中。
注意:这将允许您检查盒带是否正确锁定,或者缓冲区是否泄漏。


使用装有22G针头并充满运行缓冲液的注射器轻轻清洗孔。不要打扰井底。
添加其余的运行缓冲区。
注意:500毫升将填充一半或更少的外部水箱。


确定样品的加载顺序。在我们的说明性实验中:
 


凝胶#1(α V5印迹,以评估自我免疫共沉淀)


1)空


2)预制蛋白标准


3)mESC /unprocessing_0.75%_input                                                                                   


4)mESC / 未经处理的_IgG_ 未稀释


5)mESC的/ untreated_ α FLAG _undiluted             


6)空


7)mESC / 苯甲酸酶_0.75 %_input


8)未稀释的mESC / 苯甲酸酶_IgG


9)mESC的/ benzonase核酸_ α FLAG _undiluted             


10)空


 


Gel#2(αFLAG印迹,用于评估CoIP )


1)预制蛋白标准


2)空


3)mESC /unprocessing_0.75%_input                                                                                   


4)mESC / 未经处理的_IgG_稀释


5)mESC /未处理_αFLAG_ 稀释             


6)空


7)mESC / 苯甲酸酶_0.75 %_input


8)稀释的mESC / benzonase_IgG_


9)稀释的mESC / 苯甲酸酶_αFLAG             


10)空


注意:将蛋白质标准品上样至孔1(gel#1)或孔2(gel#2)中,以便可以轻松地区分两者。


制备预染蛋白质标准(7 微升加27 微升1×SDS上样缓冲液,17 微升每孔)。
小心地将每个样品17μl 装入P20。与17填充空孔微升1×SDS上样缓冲液中。
将运行中的录像带盒的盖子连接到电源,然后将其放在录像带盒的顶部。
在75 V下运行,直到样品进入分离凝胶。
注意:标记物碰到分离凝胶后,您会看到清晰的条带形成。


将电压增加到130V。
注意:为帮助散热,我们经常将卡带盒放在大型金属块上。


运行直到溴酚蓝碰到卡匣的底部,但要用完之后再用完。
关闭电源。
取下盖子,解锁凝胶盒并取出。
用ddH 2 O 彻底清洗盒带,包括孔,以除去运行缓冲液的任何痕迹。
将蛋白质转移到硝酸纤维素膜上
组装传输设备(图4)。
用ddH 2 O 预湿两块吸墨膜,切开仅比凝胶稍大即可转移。
注意:戴手套处理薄膜时,只能触摸薄膜的侧面。


取一个适合凝胶固定盒的托盘。
添加一个凝胶固定器盒,黑色面朝下,并倒入足够的转移缓冲液以覆盖它。
加入一块海绵,并确保将其完全浸入转印缓冲液中。
在转移缓冲液中预润湿2张吸墨纸,仅将其切成略大于要转移的凝胶。
将第一个凝胶盒放在纸巾上(向下压),然后用提供的金属刮刀将其打开。
取下顶部,确保凝胶粘附到底部。
用剃须刀刮掉伸出的凝胶底部和堆积的凝胶。
注意:堆积凝胶是胶状的。用刮刀将其从凝胶盒刮至纸巾,以清除所有残留物。


将装有凝胶的试剂盒浸入带有转移缓冲液的托盘中。
从盒中取出凝胶并将其转移到湿吸墨纸的顶部;将空纸盒放在一边。
将印迹膜在转移缓冲液中预湿,然后将其铺在凝胶上。
将一块吸水纸预湿,然后将其铺在膜的顶部。
用空的凝胶盒将凝胶和膜之间可能形成的气泡吹出,用力但要轻柔地按压。
重复步骤J3a -xii 和J3a -xiii 。
预湿另一块海绵,然后将其添加到顶部。
注意:始终预先润湿吸墨纸和海绵,以免产生毛细作用。


小心地关闭凝胶固定器盒,不要打扰三明治,然后将其插入传输槽中。
对第二个凝胶重复步骤J3a -iii 至J3a -xvi 。
 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1902918--1289 Claudia Cattoglio 809941 \ Figs jpg \ Figure4.jpg


图4.转移“三明治”的组装


 


插入冷却装置。
完全填充传输槽。
合上盖子,将电缆连接到传输电源。
在100 V和4°C下转移2小时。
阻挡膜
从转印设备中取回已弄脏的膜。
注意:如果您看到染色的分子量转移到您的膜上,您将知道转移成功。


使用足够大的容器在不弯曲的情况下快速冲洗TBS-T中的每个膜。
加入10 ml封闭液(TBS-T中含10%牛奶)。
注意:移液时要紧贴膜,不要放在膜的顶部。


在室温下搅拌至少1小时。
印迹膜
在15 ml锥形管中的印迹溶液(TBS-T中含5%的牛奶)中制备一抗。在我们的说明性实验中:
凝胶#1:兔αV5,1:2,000(8μl5 %牛奶中4μl )


凝胶#2:兔α FLAG,1:1000(8 μ 升在8ml 5%牛奶)


注意:检查所选抗体的数据表,以找到推荐的Western blot稀释液。从一种抗体到另一种抗体,这可能相差很大。


丢弃封闭溶液,并在印迹溶液中加入抗体。
在4 ℃下搅拌过夜。
注意:在冷室中过夜孵育可增加特异性信号,同时减少迄今为止我们测试的所有抗体的背景噪音。


洗膜
从膜上除去印迹溶液。
用10 ml的TBS-T快速冲洗,除去所有一抗残留物。
用新鲜的10毫升TBS-T代替,搅拌15分钟。
换上新鲜的TBS-T,搅拌5分钟。
重复步骤J6d。
用结合HRP的二抗进行印迹
在TBS-T中的5%牛奶中制备HRP 结合的二抗的1:5,000稀释液。在我们的实验说明,山羊α小鼠HRP (2 μ 升在10毫升的5%牛奶)。
在室温下搅拌至少30分钟。
按步骤J6洗涤膜。
进行化学发光反应
对于每个膜,准备2 ml ECL试剂(1 ml 氧化试剂加1 ml增强鲁米诺试剂)。
将膜转移到新的容器中,并吸出任何TBS-T残留物。
吸取ECL试剂以完全覆盖膜。
温育2分钟,轻轻旋转容器,仍要确保膜完全覆盖。
丢弃ECL试剂并成像Western blot。
注意:您可以使用X射线胶片(我们的首选)或化学发光成像仪器(例如Chemidoc )。


 


笔记


 


最重要的是,您要测试用于免疫沉淀和/或检测每个标签的抗体,以排除与其他标签以及与POI(使用未标记的野生型细胞)的交叉反应性。例如,在探查带有V5标签和带有FLAG标签的Rad21蛋白之间的自我相互作用时,我们发现其中一种FLAG抗体确实在未标记的细胞中检测到了一条具有相同大小的Rad21的条带,这表明可能与野生型Rad21 蛋白(参见图2 – Cattoglio 等人[ 2019 ]中的图补品1 )。
对于内源蛋白的标准CoIP ,您将需要确定良好的抗体以进行免疫沉淀并检测POI。如果没有可用的方法,则可以像我们对自我CoIP一样执行CRISPR / Cas9内源标签,但是这次用不同标签(例如,带有FLAG-Halo标签的CTCF和V5-在SNAP标签RAD21在克隆C59 汉森等人。[ 2019 ] )。
 


菜谱


 


储备溶液
6 N HCl(500毫升)
戴护目镜和面罩。在通风橱中工作。用250 ml的双蒸馏水(ddH 2 O)稀释250 ml的浓酸(36.5至38.0%)。


10x PBS,pH 7.4(1 升)
向800 ml的ddH 2 O中添加NaCl(80 g),KCl (2 g),Na 2 HPO 4 (14.4 g)和KH 2 PO 4 (2.4 g)。搅拌溶解
戴上护目镜,用6 N HCl调节pH值至7.4(Thermo Fisher Scientific制备6 N HCl溶液,用ddH 2 O 稀释浓酸1:1 )
用ddH 2 O 将体积调至1 L ,然后将100 ml分配到可高压灭菌的玻璃瓶中
甲上的液体循环utoclave 30分钟
1x PBS,pH 7.4
137毫米氯化钠


2.7毫米氯化钾


10毫米Na 2 HPO 4


2毫米KH 2 PO 4


稀从10倍的蒸压溶液(配方A2)和重新autoc 澡


注意:您也可以购买现成的无菌1x PBS。储存温度:15-30 ° C。保质期:从制备日期算起12个月。


1 M HEPES,pH 7.6(1 L )
从700 ml的ddH 2 O 开始。添加23 8.3 g的HEPES,使其溶解
加入31.9克KOH(防汗眼罩和面膜)
用ddH 2 O 使溶液升至定量
通过瓶到滤波器P 0.2 μ 米过滤器和等分40毫升到50ml锥形管中
储存在-20 °C长达12个月
注意:用这种方法制得的1 M溶液的pH值为〜7.8,但是将储备溶液稀释20倍后的pH值为7.6。


1 M Tris-碱pH 6.8 / 7.5(1 L )
从500 ml的ddH 2 O 开始。添加121.1 g的Tris-碱,使其溶解
用6 N HCl将pH调节至6.8 / 7.5 (戴防护眼罩和面罩)
用ddH 2 O 使溶液升至定量
甲上的液体循环utoclave 30分钟
5 M NaCl(1 升)
从700 ml的ddH 2 O 开始。添加292.2 g的NaCl并搅拌使其溶解
用ddH 2 O 使溶液升至定量
甲上的液体循环utoclave 30分钟
2 M KCl (500毫升)
从300 ml的ddH 2 O 开始。添加74. 6 g的KCl 并搅拌使其溶解
用ddH 2 O 使溶液升至定量
通过瓶顶部0.2滤波器μ 米过滤器
1 M氯化镁2 (1 升)
从800 ml的ddH 2 O 开始。添加203.3 g的MgCl 2 并搅拌使其溶解
用ddH 2 O 使溶液升至定量
甲上的液体循环utoclave 30分钟
1 M DTT(10毫升)
从8 ml的ddH 2 O 开始。添加1.54 g的DTT 并将试管倒置以溶解
用ddH 2 O 使溶液升至定量
准备1毫升等分试样,并在-20 °C下保存长达12个月
10%Triton X-100(500毫升)
向450 ml的ddH 2 O中添加50 ml的Triton X-100。


0.5 M EDTA pH 8.0(1 升)
从700 ml的ddH 2 O 开始。添加186.12 g的EDTA并搅拌(EDTA 在pH值接近8.0之前不会溶解)
用NaOH颗粒(〜20 g ;戴护目镜和口罩)将pH值调节至8.0
用ddH 2 O 使溶液升至定量
甲上的液体循环utoclave 30分钟
10%APS(10毫升)
向8 ml的ddH 2 O中添加1 g的过硫酸铵,并将试管倒置以溶解
用ddH 2 O 使溶液升至定量
准备1毫升等分试样,并在-20 °C下保存长达12个月
50x cOmplete 蛋白酶抑制剂原液(〜10 ml)
将15毫升锥形管中的10片药片溶于10毫升ddH 2 O中
倒置吨UBE直到完全溶解
准备500毫升等分试样,并在-20 °C下保存长达12个月
1 M联am(10 ml; 1,000 x )
向8 ml的ddH 2 O中加入1.2 g的苯甲idine 并将试管倒置以溶解
用ddH 2 O 使溶液升至定量
准备1毫升等分试样,并在-20 °C下保存长达12个月
0.2 M PMSF(10毫升; 1,000 x )
向9毫升异丙醇中加入348毫克PMSF ,并将试管倒置以溶解
用异丙醇将溶液升至刻度
准备1毫升等分试样,并在-20 °C下保存长达12个月
1.23 MB 为-T ris pH 6.4 / 6.7(每个250 ml)
向300 ml的ddH 2 O中添加128.7 g的Bi s-Tris,并搅拌直至溶解
将容量调至最大400毫升,并平均分成两个烧杯(每个200毫升)
用浓盐酸将pH调至6.4或6.7 (戴防护眼罩和面罩)
用ddH 2 O 将每种溶液加至250 ml
过滤器灭菌并在室温下储存长达12个月
 


缓冲液和其他解决方案
细胞裂解缓冲液(10 ml)[最终浓度]:
100 μ 升的1M HEPES的,pH值7.9 [10 mm]的


50 微升的2M的氯化钾[10 mm]的


30 微升的1M的的MgCl 2 [3 mm]的


1.16克蔗糖[〜340毫米]


1毫升甘油[10%]


10 微升的1 M DTT [1 mm]的


ddH 2 O至10 ml


称重蔗糖并添加〜5 ml的ddH 2 O
加入所有剩余的溶液,并用更多的ddH 2 O 使体积达到10 ml
用注射器过滤通过0.2 μ 米过滤器
右在使用之前,添加的Triton X-100至0.1%的最终(使用10%的Triton X-100的库存为100 X 浓溶液)和preotease 抑制剂(抑肽酶,苯甲脒,PMSF和大鹏他蛋白酶抑制剂混合物)
0.1 M CoIP 缓冲液(500 ml)[最终浓度]
10毫升5 M氯化钠[100毫米]


12.5毫升的1 M Hepes pH 7.9 [25 mM]


500 微升的1M氯化镁的2 [1 mm]的


200 微升的0.5M的EDTA pH为8.0 [0.2 mm]的


25毫升10%NP-40替代品[0.5%]


ddH 2 O至500 ml


向100 ml的ddH 2 O中添加所有其他溶液。使体积至500ml,并过滤通过一个瓶顶部0.2 μ 米过滤器


0.2 M CoIP 缓冲液(500 ml)[最终浓度]
20 ml的5 M NaCl [200 mM]


12.5毫升1 M HEPES pH 7.9 [25 mM]


500 微升的1M氯化镁的2 [1 mm]的


200 微升的0.5M的EDTA pH为8.0 [0.2 mm]的


25毫升10%NP-40替代品[0.5%]


ddH 2 O至500 ml


向100 ml的ddH 2 O中添加所有其他溶液。使体积至500ml,并过滤THR ough一个瓶顶部0.2 微米过滤器


在0.2 M CoIP 缓冲液(50 ml)中的0.5%BSA
向50 ml的0.2 M CoIP 缓冲液中加入250 mg的BSA。颠倒管子进行混合,直到BSA溶解,然后通过5 0 ml离心管顶部过滤器过滤


20x MOPS运行缓冲区(1 L )
209.39克MOPS


121.1克Tris-Base


20克SDS(戴防护眼罩和口罩)


7.44克EDTA


使体积达到1升的DDH 2 O和过滤器通过瓶顶部0.2 μ 米过滤器
在室温下避光保存长达12个月
要运行SDS-PAGE,请用ddH 2 O 稀释至1x,然后添加偏亚硫酸氢钠(每500 ml运行缓冲液0.47 5 g)
每次准备新鲜的1x运行缓冲液;不要重复使用
TR ansfer缓冲液(2 大号)
3.63克Tris-Base


28.83克甘氨酸


400毫升甲醇


将Tris-Base和甘氨酸溶解在1升ddH 2 O中
将体积调至1.6升并添加甲醇
在4°C下保存最多12个月。我们通常会在丢弃同一个传输缓冲区之前重复使用两次
TBS-T(2 升)
200毫升5 M氯化钠


10毫升2 M Tris pH 7.5


2毫升的吐温20


向1.5升ddH 2 O中添加NaCl和Tris
将容量升至2升,转移到干净的瓶子中,添加磁力搅拌器和Tween-20(我们通常使用P1000,将充满Tween-20 的整个吸头滴入瓶中)
搅拌,直到所有的Tween-20的被均匀地通过瓶顶部0.2分布和过滤器μ 米过滤器
4x样品缓冲液(10毫升)[最终浓度]
1.6毫升2-巯基乙醇[16%]


2 ml Tris pH 6.8 [200 mM]


0.8克SDS(戴防护眼罩和面罩)[8%]


4毫升甘油[40%]


0.62克DTT [400毫米]


0.04克溴酚蓝[0.4%]


称量所有成分在50毫升锥形管中的重量
通过0.2滤波器μ 米滤波器50毫升过滤管
对于2x样品缓冲液,用ddH 2 O 1:1稀释4x溶液


对于1x样品缓冲液,将2x溶液用ddH 2 O 1:1稀释。准备1 ml等分试样,并在-20°C下保存长达12个月


封闭溶液,含10%牛奶(w / v)的TBS-T(100 ml)
将10克速溶牛奶溶于100毫升TBS-T。在4 °C下保存最多2周


印迹溶液,5%牛奶(w / v)的TBS-T(100 ml)
将5克速溶牛奶溶于100毫升TBS-T。在4 °C下保存最多2周


 


致谢


 


ASH感谢Siebel干细胞研究所的博士后研究金和NIH NIGMS K99独立之路K99GM130896的支持。该泽南-Darzacq 实验室是由美国国立卫生研究院共同基金4D支持Nucleome 计划U01-EB021236和U54-DK107980(XD),再生医学批LA1-08013加州理工学院(XD),以及由霍华德·休斯医学研究所(003061,RT )。我们首先开发了此处详述的协议,以评估CTCF (Hansen 等人,2019)和黏附素亚基Rad21(Cattoglio 等人,2019)的自我相互作用。感谢李杰(Jack Li)对这份手稿提供意见。


 


利益争夺


 


作者宣称没有任何竞争性的金融/非金融利益。


 


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Copyright Cattoglio et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Cattoglio, C., Pustova, I., Darzacq, X., Tjian, R. and Hansen, A. S. (2020). Assessing Self-interaction of Mammalian Nuclear Proteins by Co-immunoprecipitation. Bio-protocol 10(4): e3526. DOI: 10.21769/BioProtoc.3526.
  2. Cattoglio, C., Pustova, I., Walther, N., Ho, J. J., Hantsche-Grininger, M., Inouye, C. J., Hossain, M. J., Dailey, G. M., Ellenberg, J., Darzacq, X., Tjian, R. and Hansen, A. S. (2019). Determining cellular CTCF and cohesin abundances to constrain 3D genome models. Elife 8: e40164.
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