参见作者原研究论文

本实验方案简略版
May 2016

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MPM-2 Mediated Immunoprecipitation of Proteins Undergoing Proline-directed Phosphorylation
脯氨酸依赖性磷酸化蛋白质的MPM-2介导的免疫沉淀   

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Abstract

Immunoprecipitation (IP) represents a widely utilized biochemical method to isolate a specific protein from a complex mixture taking advantage of an antibody that specifically recognizes that particular target molecule. This procedure is extremely versatile and can be applied to concentrate a specific protein, to identify interacting partners in complex with it or to detect post-translational modifications. The mitotic protein monoclonal 2 (MPM-2) is an antibody originally raised against extracts of synchronized mitotic HeLa cells to identify proteins selectively present in mitotic, and not in interphase-cells (Davis et al., 1983). MPM-2 recognizes phosphorylated serine or threonine residues followed by proline (pS/T-P), consensus epitopes generated by the concerted action of proline-directed protein kinases and phosphatases (Lu et al., 2002). These reversible phosphorylation events have emerged to control various cellular processes by promoting conformational changes on the target that are not simply due to the phosphorylation event per se. These motifs, once phosphorylated, are able to recruit Pin1 (Peptidyl-prolyl Isomerase NIMA interacting protein 1) (Lu et al., 1996; Lu and Zhou, 2007), a chaperone which drives the cis/trans isomerization reaction on the peptide bond, switching the substrate between functionally diverse conformations (Lu, 2004; Wulf et al., 2005). This protocol describes a general MPM-2 based immunoprecipitation strategy using the scaffolding molecule postsynaptic density protein-95 (PSD-95) (Chen et al., 2005), a neuronal Pin1 target (Antonelli et al., 2016), as an example to illustrate the detailed procedure.

Keywords: MPM-2 antibody (MPM-2抗体), Proline-directed phosphorylation (脯氨酸依赖性磷酸化), Peptidyl-prolyl isomerase Pin1 (肽脯氨酰异构酶Pin1), Immunoprecipitation (免疫沉淀), Scaffolding molecules (支架分子)

Background

Identification of antigens recognized by MPM-2 antibody represents a useful starting point for the discovery of target molecules undergoing post-phosphorylation prolyl-isomerization regulatory mechanism. The prolyl isomerase Pin1, in fact, shares with MPM-2 antibody the same recognition motif as well as the phospho-dependency of the binding. The main advantage of using the MPM-2 antibody in immunoprecipitation experiments is the achievement of a selective enrichment of the phosphorylated targets over the unphosphorylated counterparts, which are frequently present in much greater quantities in the cell. Precipitated antigens by virtue of such post-translational modification can be then easily identified by standard western blotting using highly specific primary antibodies.

Materials and Reagents

  1. 10 cm2 Petri dishes (Corning, catalog number: 353003 )
  2. Dissection plate (Sigma-Aldrich, catalog number: P7741 )
  3. 1.5 ml reaction tube (SARSTEDT, catalog number: 72.706.700 )
  4. Surgical scalpel blade number 11 (Sigma-Aldrich, catalog number: S2771 )
  5. 26 gauge needle (BD, PrecisionGlideTM, catalog number: 305110 )
  6. Amersham Protran Premium 0.2 NC (GE Healthcare, catalog number: 10600004 )
  7. HEK293 cells
  8. C57BL/6 mouse strain (or any other mouse model, both genders)
  9. pAcGFP1-C1 vector (Takara Bio, Clontech, catalog number: 632470 )
  10. FLAG-PSD-95 plasmid DNA (Craven et al., 1999)
  11. Dulbecco’s modified Eagle’s medium (DMEM) high glucose, GlutaMAXTM supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 10566016 )
  12. 10% fetal bovine serum (FBS)
  13. Polyethylenimine, Linear (PEI) (Polysciences, catalog number: 23966-2 )
  14. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140148 )
  15. cOmpleteTM EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 11873580001 )
  16. Phosphatase inhibitor cocktail 1 (Sigma-Aldrich, catalog number: P2850 )
  17. IgG from mouse serum (Sigma-Aldrich, catalog number: I8765 )
  18. Anti-FLAG (clone M2) (Sigma-Aldrich, catalog number: F9291 )
  19. Phospho-Serine/Threonine-Proline MPM2 antibody (EMD Millipore, catalog number: 05-368 )
  20. Protein G Sepharose 4 Fast Flow (GE Healthcare, catalog number: 17-0618-01 )
  21. PageRulerTM prestained protein ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 26616 )
  22. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  23. Goat anti-mouse HRP conjugated IgG (EMD Millipore, catalog number: 12-349 )
  24. ECL detection reagents (GE Healthcare, catalog number: RPN2209 )
  25. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  26. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  27. Disodium hydrogen phosphate (Na2HPO4) (Sigma-Aldrich, catalog number: 7558-79-4 )
  28. Potassium dihydrogen phosphate (KH2PO4) (Sigma-Aldrich, catalog number: 7778-77-0 )
  29. Tween 20 (Sigma-Aldrich, catalog number: P9416 )
  30. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  31. NP-40 (Sigma-Aldrich, catalog number: I3021 )
  32. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  33. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  34. Sodium orthovanadate (Na3VO4) (Sigma-Aldrich, catalog number: S6508 )
  35. Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: 201154 )
  36. TEMED (Sigma-Aldrich, catalog number: T9281 )
  37. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  38. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  39. Methanol (Sigma-Aldrich, catalog number: 322415 )
  40. Glycine (Sigma-Aldrich, catalog number: G8898 )
  41. Acrylamide/bis-acrylamide (29:1) (Sigma-Aldrich, catalog number: A2792 )
  42. Ammonium persulfate (Sigma-Aldrich, catalog number: A3678 )
  43. Phosphate buffered saline (1x PBS) (see Recipes)
  44. MPM-2 lysis buffer (see Recipes)
  45. Phosphatase inhibitors (see Recipes)
  46. IP wash buffer (see Recipes)
  47. 2x Laemmli sample buffer (see Recipes)
  48. Western blot running buffer (see Recipes)
  49. Western blot transfer buffer (see Recipes)
  50. Tris-buffered saline-Tween 20 (TBS-T) (see Recipes)
  51. BSA blocking solution (see Recipes)

Equipment

  1. Nikon Diaphon 300 phase contrast inverted tissue culture microscope
  2. Cell scraper (Sigma-Aldrich, catalog number: C5981 )
  3. Refrigerated centrifuge (Eppendorf, model: 5424R )
  4. Incubator (Panasonic Biomedical Sales Europe, model: MCO-18AC-PE )
  5. DNA sonicator (EpiShear, model: Cooled Sonication Platform )
  6. Rotator mixer (Scilogex, model: MX-RD-Pro LCD Digital Tube Rotator Mixer )
  7. Tissue homogenizer (Thomas Scientific, catalog number: 3409Y72 )
  8. Cold room, 4 °C
  9. Polyacrylamide gel electrophoresis system (Bio-Rad Laboratories, catalog number: 1658000EDU )
  10. Western blot transfer apparatus (Bio-Rad Laboratories, model : Trans-Blot® Cell )

Software

  1. Alliance 4.7 software (UVITECH)
  2. UVI band Imager software

Procedure

  1. FLAG-PSD-95 ectopically expressed in HEK293
    1. HEK293 cells are cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% 100 μg/ml penicillin and 100 μg/ml streptomycin.
    2. 10 cm2 Petri dishes are transfected with 2 μg FLAG-tagged PSD-95 using PEI transfection method. In particular, 1.8 x 106 cells are plated the day before transfection. Total plasmid DNA is diluted in 500 μl serum-free DMEM (w/o) phenol red. PEI (1 μg/μl) is then added to the diluted DNA on a 7:1 ratio (μg PEI:μg DNA). The mixture is incubated for 20 min at RT and added dropwise. Cells are harvested 48 h post-transfection. Transfection efficiency is evaluated by simultaneous co-transfection of 500 ng of pGFP-C1, a vector DNA encoding the green fluorescent protein. Cells expressing GFP are directly visualized by fluorescence microscopy.
    3. Remove the medium and wash two times cell culture dishes with ice-cold PBS.
    4. Scrape adherent cells off the dish using a cell scraper, and then gently transfer the cell suspension into a fresh pre-cooled 1.5 ml reaction tube.
    5. Centrifuge at 13,500 x g for 10 min at 4 °C (refrigerated centrifuge). Discard the supernatant and collect the pellet.
    6. Mechanically resuspend the pellet in 700 μl of MPM-2 lysis buffer supplemented with protease (cOmpleteTM EDTA-free inhibitor cocktail) and phosphatase inhibitors (see Recipes).
    7. Incubate lysates for 30 min at 4 °C with rotation.
    8. Sonicate lysates with three short bursts of 10 sec each followed by intervals of 30 sec for cooling. Keep lysates at all times on ice and avoid foaming.
      Note: Sonication is mainly performed to shear DNA/RNA, making the lysate progressively less viscous.
    9. Centrifuge at 13,500 x g for 10 min at 4 °C (refrigerated centrifuge).
    10. Pipette out supernatant into a fresh reaction tube and discard the pellets.

  2. Endogenous PSD-95 from mouse brain
    1. Sacrifice mice according to the Italian Animal Welfare legislation (D.L. 26/2014) that implements the European Committee Council Directive (2010/63 EEC). The procedures are approved by local veterinary authorities and by the ethical committee of SISSA. After CO2 exposure with the purpose of inducing unconsciousness, postnatal day (P) 10-P15 animals (male and female) are decapitated with sharp surgical scissors. The brain is quickly removed from the skull, rinsed in ice-cold PBS, transferred to an ice-cold dissection plate and minced using a surgical scalpel blade (number 11). Broken up tissue is homogenized at 4 °C using tissue homogenizer, setting 500 rpm 10 times, in MPM-2 lysis buffer supplemented with protease and phosphatase inhibitors (as above). For each 50 mg of brain tissue is added 400 µl of lysis buffer to obtain an optimal protein concentration ranging from 10 to 20 mg/ml. For a P15 mouse brain weighing around 300-350 mg are added 3.0 ml of lysis buffer.
    2. Incubate tissue homogenate for 30 min-1 h at 4 °C with rotation.
    3. Sonicate as previously described and pass several times through a 26 gauge needle the homogenate.
    4. Centrifuge at 13,500 x g for 10 min at 4 °C.
    5. Gently remove the reaction tubes from the centrifuge, pipette out the supernatant and divide it into two 1.5 ml fresh reaction tubes pre-cooled on ice; discard the pellets.

  3. Phospho-Serine/Threonine-Proline immunoprecipitation
    1. Save 30 μl of cell lysate or homogenate as starting material (input) to be tested in SDS-PAGE analysis and 5.0 μl to determine the protein concentration.
    2. Split the remaining lysate equally into two fresh reaction tubes for MPM-2 immunoprecipitation and normal mouse IgG as control sample (around 1-1.5 mg of total protein in each tube).
    3. Add 2 μg of MPM-2 antibody and 2 μg of control mouse IgG to the appropriate reaction tube.
      Note: The appropriate ratio of MPM-2 antibody/total protein input has to be titrated in preliminary experiments, taking into account the level of expression of the protein under investigation and the number of putative pS/pT-P consensus motifs present on it.
    4. Incubate 3 h at 4 °C with gentle rotation.
      Note: The incubation time with MPM-2 antibody follows the same rationale discussed above (step C3). If the protein of interest is poorly expressed overnight incubation is recommended.
    5. Add 100 μl (50% beads/buffer) of Protein-G Sepharose beads equilibrated into MPM-2 lysis buffer to each sample.
      Note: To equilibrate the beads, add MPM-2 lysis buffer to the bead, mix in the tube several times, spin down the beads, and remove the supernatant (done twice).
    6. Pull-down the antibody-antigen complexes bound to Protein-G Sepharose beads by incubating for further 2 h at 4 °C with gentle rotation.
    7. Wash beads three times with lysis buffer.
      Note: For wash beads, add buffer to the beads, mix the tube several times, spin down the beads, and remove the supernatant.
    8. After the last wash, pipette out the supernatant and elute proteins by heating to 95-100 °C for 5 min with 50 µl of 2x Laemmli sample buffer. Samples can be immediately loaded onto the gel or kept frozen at -80 °C to be run later.
    9. Analyze samples using SDS-PAGE and Western blotting.
      Load 20 μg of starting material (Input), 50 μl of the IP samples and 7 μl of prestained protein ladder onto SDS-PAGE gel (10 x 10 cm).
      Note: In case of MPM-2 detection in Western blot analysis a 2 h transfer is recommended to minimize or avoid loss of post-translational modifications.
    10. Detection of the immunoprecipitated protein using either the target specific antibody or the MPM-2 antibody.
      Note: For MPM2 detection block the nitrocellulose membrane with 2.5% BSA in PBS-T (see Recipes) instead of 5% nonfat milk in TBS-T used in standard conditions. Incubate the membrane with MPM-2 antibody (1:500 dilution in BSA Blocking solution) with gentle agitation either overnight at 4 °C or 2 h at RT. After three washes of 5 min each with BSA blocking solution incubate the membrane with HRP-conjugated goat anti-rabbit secondary antibody (1:1,000 dilution in BSA blocking solution) with gentle agitation for 1 h at RT.
    11. Western blot image acquisition is performed using the ECL detection reagents (according to the manufacturer’s protocol) and the Alliance 4.7 software.
      Note: In case of aspecific binding to control IgG lysates, a pre-clearing step should be performed. To each sample add 100 μl (50% beads/buffer) of Protein G Sepharose beads and incubate 1 h with gentle rotation at 4 °C. Spin down the beads and collect the supernatants. Add the immunoprecipitating antibodies as described in step C3.

Data analysis

Quantification of immunoprecipitated target protein was determined by densitometry analysis on the acquired images with Alliance 4.7 software (UVITECH). The volume of the bands corresponding to Input (1/20 of the total lysate) and the immunoprecipitated fraction were determined using the UVIband imager software (Amersham). The amount of immunoprecipitated is normalized to the corresponding inputs.

Representative data

Results from a representative experiment are shown in Figure 1.


Figure 1. MPM-2 mediated immunoprecipitation of PSD-95 either ectopically expressed in HEK-293 cells or endogenously present in mouse brain. A. FLAG-PSD-95 ectopically expressed in HEK-293 cells was immunoprecipitated with anti-MPM-2 antibody. Each sample was run twice to perform immunoblots with anti-FLAG and anti MPM2 antibodies, as indicated. Mouse IgG was used as negative control. B. PSD-95 was precipitated from brain extracts and unveiled in Western blot using the anti-MPM2 antibody. Asterisks indicate background bands due to secondary antibody cross-reactivity with IgG heavy chains used in immunoprecipitation.
Note: Only a fraction of PSD-95 is phosphorylated at S-T/P consensus motifs (compare Input lanes).

Recipes

  1. Phosphate buffered saline (1x PBS)
    137 mM NaCl
    2.7 mM KCl
    4.3 mM Na2HPO4
    1.47 mM KH2PO4
    Adjust to a final pH of 7.4
    Note: In PBS-T add 0.1% Tween 20.
  2. MPM-2 lysis buffer
    50 mM Tris-HCl (pH 7.5)
    1% NP-40
    0.5% Triton X-100
    150 mM NaCl
    10% glycerol
    10 mM sodium orthovanadate
    1 mM sodium fluoride
    1% phosphatase inhibitor cocktail 1
    1x cOmpleteTM EDTA-free protease inhibitor cocktail
  3. Phosphatase inhibitors
    1 mM sodium orthovanadate
    1 mM sodium fluoride
    Phosphatase inhibitor cocktail 1
  4. IP wash buffer
    50 mM Tris-HCl (pH 7.5)
    100 mM NaCl
    0.1% Tween 20
    0.1% SDS
    10 mM sodium orthovanadate
    1 mM sodium fluoride
    1% phosphatase inhibitor cocktail 1
    1x cOmpleteTM EDTA-free protease inhibitor cocktail
  5. 2x Laemmli sample buffer
    100 mM Tris-HCl (pH 6.8)
    4% SDS
    20% glycerol
    1% 2-mercaptoethanol
    0.001% bromophenol blue
  6. Western blot running buffer
    25 mM Tris
    190 mM glycine
    0.1% SDS
  7. Western blot transfer buffer
    25 mM Tris (pH 8.3)
    192 mM glycine
    10% methanol
    0.1% SDS
  8. Tris-buffered saline-Tween 20 (1x TBS-T)
    50 mM Tris (pH 7.6)
    150 mM NaCl
    0.1% Tween 20
  9. BSA blocking solution (used also as antibody incubation buffer)
    1x TBS-T
    2.5% BSA

Note: All buffers are stored at RT except the BSA blocking solution which is kept at 4 °C (no longer than a week). Sample buffer is stored without 2-mercaptoethanol which is freshly added before use. Only buffer’s volumes required for the immunoprecipitation procedure are pre-cooled on ice and supplied with protease and phosphatase inhibitors just before use. All inhibitors are resuspended in sterilized MilliQ water, aliquoted and stored at -20 °C. Sodium orthovanadate is prepared as 200 mM stock solution in sterilized MilliQ water, adjusting the pH to 10.0. At this pH the solution is yellow. To ensure the presence of vanadate monomers the solution is boiled until translucent and the pH is readjusted to 10.0.

Acknowledgments

We are grateful to Dr. S. Vicini (Georgetown University School of Medicine, Washington D.C., USA), for kindly providing PSD-95 cDNA. This work was supported by grants from the Beneficentia Stiftung (BEN 2014/08) and from Telethon (GGP11043).

References

  1. Antonelli, R., De Filippo, R., Middei, S., Stancheva, S., Pastore, B., Ammassari-Teule, M., Barberis, A., Cherubini, E. and Zacchi, P. (2016). Pin1 modulates the synaptic content of NMDA receptors via prolyl-isomerization of PSD-95. J Neurosci 36(20): 5437-5447.
  2. Chen, X., Vinade, L., Leapman, R. D., Petersen, J. D., Nakagawa, T., Phillips, T. M., Sheng, M. and Reese, T. S. (2005). Mass of the postsynaptic density and enumeration of three key molecules. Proc Natl Acad Sci U S A 102(32): 11551-11556.
  3. Craven, S. E., El-Husseini, A. E. and Bredt, D. S. (1999). Synaptic targeting of the postsynaptic density protein PSD-95 mediated by lipid and protein motifs. Neuron 22(3): 497-509.
  4. Davis, F. M., Tsao, T. Y., Fowler, S. K. and Rao, P. N. (1983). Monoclonal antibodies to mitotic cells. Proc Natl Acad Sci U S A 80(10): 2926-2930.
  5. Lu, K. P. (2004). Pinning down cell signaling, cancer and Alzheimer's disease. Trends Biochem Sci 29(4): 200-209.
  6. Lu, K. P., Hanes, S. D. and Hunter, T. (1996). A human peptidyl-prolyl isomerase essential for regulation of mitosis. Nature 380(6574): 544-547.
  7. Lu, K. P., Liou, Y. C. and Zhou, X. Z. (2002). Pinning down proline-directed phosphorylation signaling. Trends Cell Biol 12(4): 164-172.
  8. Lu, K. P. and Zhou, X. Z. (2007). The prolyl isomerase PIN1: a pivotal new twist in phosphorylation signalling and disease. Nat Rev Mol Cell Biol 8(11): 904-916.
  9. Wulf, G., Finn, G., Suizu, F. and Lu, K. P. (2005). Phosphorylation-specific prolyl isomerization: is there an underlying theme? Nat Cell Biol 7(5): 435-441.

简介

免疫沉淀(IP)代表广泛使用的生物化学方法,以利用特异性识别特定靶分子的抗体从复杂混合物中分离特异性蛋白质。该程序是非常通用的,可以应用于集中特定蛋白质,识别与其复合的相互作用的配偶体或检测翻译后修饰。有丝分裂蛋白单克隆2(MPM-2)是最初针对同步有丝分裂HeLa细胞的提取物产生的抗体,以鉴定选择性存在于有丝分裂中的蛋白质,而不是在间期细胞中(Davis等,1983)。 MPM-2识别磷酸化的丝氨酸或苏氨酸残基,随后是脯氨酸(pS / T-P),由脯氨酸指导的蛋白激酶和磷酸酶的共同作用产生的共有表位(Lu等,2002)。这些可逆磷酸化事件已经出现以通过促进目标上的构象变化来控制各种细胞过程,这不仅仅是由于磷酸化事件本身。这些基序一旦被磷酸化,就能够招募Pin1(肽基 - 脯氨酰异构酶NIMA相互作用蛋白1)(Lu等人,1996; Lu和Zhou,2007),这是一个促进肽键上的顺式/反式异构化反应的伴侣,在基础功能不同的构象之间切换底层(Lu,2004; Wulf等人,2005)。该方案描述了使用支架分子突触后密度蛋白-95(PSD-95)(Chen等人,2005),神经元Pin1靶(Antonelli等,2016)作为示例的基于MPM-2的免疫沉淀策略以说明详细的程序。
【背景】由MPM-2抗体识别的抗原的鉴定代表了发现经过磷酸化脯氨酰异构化调控机制的靶分子的有用起点。 事实上,脯氨酰异构酶Pin1与MPM-2抗体共享相同的识别基序以及结合的磷酸依赖性。 在免疫沉淀实验中使用MPM-2抗体的主要优点是在非磷酸化对应物上实现磷酸化靶标的选择性富集,其在细胞中经常以更大的量存在。 通过这种翻译后修饰的沉淀抗原可以在使用高度特异性一级抗体的标准蛋白质印迹中容易地鉴定。

关键字:MPM-2抗体, 脯氨酸依赖性磷酸化, 肽脯氨酰异构酶Pin1, 免疫沉淀, 支架分子

材料和试剂

  1. 10cm 培养皿(Corning,目录号:353003)
  2. 解剖板(Sigma-Aldrich,目录号:P7741)
  3. 1.5ml反应管(SARSTEDT,目录号:72.706.700)
  4. 外科手术刀刀片号11(Sigma-Aldrich,目录号:S2771)
  5. 26号针(BD,PrecisionGlide TM ,目录号:305110)
  6. Amersham Protran Premium 0.2 NC(GE Healthcare,目录号:10600004)
  7. HEK293细胞
  8. C57BL/6小鼠品系(或任何其他小鼠模型,两种性别)
  9. pAcGFP1-C1载体(Takara Bio,Clontech,目录号:632470)
  10. FLAG-PSD-95质粒DNA(Craven等人,1999)
  11. Dulbecco's改良的Eagle培养基(DMEM)高葡萄糖,GlutaMAX补充剂(Thermo Fisher Scientific,Gibco TM ,目录号:10566016)
  12. 10%胎牛血清(FBS)
  13. 聚乙烯亚胺,线性(PEI)(Polysciences,目录号:23966-2)
  14. 青霉素 - 链霉素(10,000U/ml)(Thermo Fisher Scientific,Gibco TM ,目录号:15140148)
  15. 不含EDTA的蛋白酶抑制剂混合物片剂(Roche Diagnostics,目录号:11873580001)
  16. 磷酸酶抑制剂混合物1(Sigma-Aldrich,目录号:P2850)
  17. 来自小鼠血清的IgG(Sigma-Aldrich,目录号:I8765)
  18. 抗FLAG(克隆M2)(Sigma-Aldrich,目录号:F9291)
  19. 磷酸 - 丝氨酸/苏氨酸 - 脯氨酸MPM2抗体(EMD Millipore,目录号:05-368)
  20. 蛋白G Sepharose 4 Fast Flow(GE Healthcare,目录号:17-0618-01)
  21. PageRuler TM 预染蛋白梯(Thermo Fisher scientific,Thermo scientific TM ,目录号:26616)
  22. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
  23. 山羊抗小鼠HRP缀合的IgG(EMD Millipore,目录号:12-349)
  24. ECL检测试剂(GE Healthcare,目录号:RPN2209)
  25. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  26. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  27. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:7558-79-4)
  28. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:7778-77-0)
  29. 吐温20(Sigma-Aldrich,目录号:P9416)
  30. (Sigma-Aldrich,目录号:T1503)
  31. NP-40(Sigma-Aldrich,目录号:I3021)
  32. Triton X-100(Sigma-Aldrich,目录号:T8787)
  33. 甘油(Sigma-Aldrich,目录号:G5516)
  34. 原钒酸钠(Na 3 VO 4)(Sigma-Aldrich,目录号:S6508)
  35. 氟化钠(NaF)(Sigma-Aldrich,目录号:201154)
  36. TEMED(Sigma-Aldrich,目录号:T9281)
  37. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  38. 溴酚蓝(Sigma-Aldrich,目录号:B0126)
  39. 甲醇(Sigma-Aldrich,目录号:322415)
  40. 甘氨酸(Sigma-Aldrich,目录号:G8898)
  41. 丙烯酰胺/双丙烯酰胺(29:1)(Sigma-Aldrich,目录号:A2792)
  42. 过硫酸铵(Sigma-Aldrich,目录号:A3678)
  43. 磷酸盐缓冲盐水(1x PBS)(参见配方)
  44. MPM-2裂解缓冲液(参见配方)
  45. 磷酸酶抑制剂(参见配方)
  46. IP洗涤缓冲液(参见配方)
  47. 2x Laemmli样品缓冲液(参见配方)
  48. 蛋白质印迹运行缓冲液(参见配方)
  49. Western印迹转移缓冲液(参见配方)
  50. Tris缓冲盐水-Tween 20(TBS-T)(参见Recipes)
  51. BSA封闭溶液(见配方)

设备

  1. 尼康Diaphon 300相位倒置组织培养显微镜
  2. 细胞刮刀(Sigma-Aldrich,目录号:C5981)
  3. 冷冻离心机(Eppendorf,型号:5424R)
  4. 孵化器(Panasonic Biomedical Sales Europe,型号:MCO-18AC-PE)
  5. DNA超声仪(EpiShear,型号:冷却超声平台)
  6. 旋转混合器(Scilogex,型号:MX-RD-Pro LCD数字管旋转混合器)
  7. 组织匀浆器(Thomas Scientific,目录号:3409Y72)
  8. 冷室,4°C
  9. 聚丙烯酰胺凝胶电泳系统(Bio-Rad Laboratories,目录号:1658000EDU)
  10. Western印迹转移装置(Bio-Rad Laboratories,型号:Trans-Blot Cell)

软件

  1. Alliance 4.7软件(UVITECH)
  2. UVI波段成像仪软件

程序

  1. FLAG-PSD-95在HEK293中异位表达
    1. 将HEK293细胞在补充有10%胎牛血清(FBS)和1%100μg/ml青霉素和100μg/ml链霉素的Dulbecco改良的Eagle培养基(DMEM)中培养。
    2. 10cm 2培养皿用2μgFLAG标记的PSD-95使用PEI转染方法转染。特别地,在转染前一天铺板1.8×10 6个细胞。将总质粒DNA在500μl无血清DMEM(w/o)酚红中稀释。然后以7:1的比例(μgPEI:μgDNA)将PEI(1μg/μl)加入到稀释的DNA中。将混合物在室温下温育20分钟并逐滴加入。转染后48小时收获细胞。通过同时共转染500ng pGFP-C1(编码绿色荧光蛋白的载体DNA)来评估转染效率。表达GFP的细胞通过荧光显微镜直接观察。
    3. 取出培养基,用冰冷的PBS洗涤两次细胞培养皿
    4. 使用细胞刮刀从培养皿上刮下贴壁细胞,然后将细胞悬浮液轻轻转移到新鲜的预冷却的1.5ml反应管中。
    5. 在4℃下以13,500×g离心10分钟(冷冻离心机)。弃去上清液并收集沉淀。
    6. 机械地将沉淀物重悬在补充有蛋白酶(cOmplete TM无EDTA抑制剂混合物)和磷酸酶抑制剂(参见Recipes)的700μlMPM-2裂解缓冲液中。
    7. 孵育裂解物30分钟,在4℃下旋转
    8. 超声裂解物,每次10秒的三次短脉冲,随后30秒的间隔冷却。保持裂解物在任何时候在冰上,避免起泡 注意:超声处理主要用于剪切DNA/RNA,使裂解液粘度逐渐降低。
    9. 在4℃下以13,500×g离心10分钟(冷冻离心机)。
    10. 将上清液移入新鲜的反应管中,弃去沉淀
  2. 内源性PSD-95从小鼠脑
    1. 根据实施欧洲委员会理事会指令(2010/63 EEC)的意大利动物福利立法(D.L. 26/2014)牺牲小鼠。这些程序由当地兽医当局和SISSA的伦理委员会批准。在诱导无意识的CO 2暴露后,用锋利的外科剪刀将产后天(P)10-P15动物(雄性和雌性)断头。将脑从颅骨中快速移出,在冰冷的PBS中冲洗,转移至冰冷的解剖板,并使用外科手术刀刀片(11号)切碎。将破碎的组织在4℃下使用组织匀浆器匀浆化,在补充有蛋白酶和磷酸酶抑制剂(如上)的MPM-2裂解缓冲液中设置500rpm 10次。对于每50mg脑组织,加入400μl裂解缓冲液以获得10至20mg/ml的最佳蛋白质浓度。对于体重约300-350mg的P15小鼠脑,加入3.0ml裂解缓冲液
    2. 孵育组织匀浆30分钟-1小时在4°C与旋转。
    3. 如前所述进行超声处理,并通过26号针均匀地通过几次
    4. 在4℃下以13,500xg离心10分钟。
    5. 轻轻地从离心机中取出反应管,移出上清液,并将其分成两个1.5ml在冰上预冷却的新鲜反应管;丢弃颗粒。

  3. 磷酸 - 丝氨酸/苏氨酸 - 脯氨酸免疫沉淀
    1. 保存30μl的细胞裂解液或匀浆作为起始材料(输入)在SDS-PAGE分析中测试和5.0μl,以确定蛋白质浓度。
    2. 将剩余的裂解物等量分成用于MPM-2免疫沉淀的两个新鲜反应管和作为对照样品的正常小鼠IgG(每个管中约1-1.5mg总蛋白)。
    3. 向适当的反应管中加入2μg的MPM-2抗体和2μg的对照小鼠IgG。
      注意:在初步实验中必须滴定MPM-2抗体/总蛋白输入的适当比例,考虑到所研究的蛋白质的表达水平和存在的假定的pS/pT-P共有基序的数目
    4. 在4℃下温和旋转孵育3小时 注意:与MPM-2抗体的孵育时间遵循上述相同的理论(步骤C3)。如果目的蛋白质表达不佳,建议过夜孵育。
    5. 向每个样品中加入100μl(50%珠/缓冲液)平衡到MPM-2裂解缓冲液中的Protein-G Sepharose珠。
      注意:为了平衡珠子,向珠子中加入MPM-2裂解缓冲液,在管中混合数次,旋转珠子,除去上清液(两次)。
    6. 通过在4℃下温和旋转孵育另外2小时,下拉结合到蛋白-G琼脂糖珠上的抗体 - 抗原复合物。
    7. 用裂解缓冲液洗涤珠子三次。
      注意:对于洗涤珠,向珠中加入缓冲液,混合管数次,旋下珠,并除去上清液。
    8. 最后一次洗涤后,吸出上清液,通过加热至95-100℃5分钟,用50μl2×Laemmli样品缓冲液洗脱蛋白质。样品可以立即加载到凝胶上或保存在-80℃冷冻以备以后运行。
    9. 使用SDS-PAGE和Western印迹分析样品。
      将20μg起始材料(输入),50μlIP样品和7μl预染色的蛋白梯度加载到SDS-PAGE凝胶(10×10cm)上。
      注意:在Western印迹分析中检测MPM-2的情况下,建议进行2小时转移,以尽量减少或避免翻译后修饰的丢失。
    10. 使用靶特异性抗体或MPM-2抗体检测免疫沉淀的蛋白质 注意:对于MPM2检测,用在PBS-T中的2.5%BSA(参见Recipes)代替在标准条件下使用的TBS-T中的5%脱脂乳封闭硝酸纤维素膜。用MPM-2抗体(BSA封闭溶液中的1:500稀释液)孵育膜,在4℃下温和搅拌过夜或在室温下温育2小时。在用BSA封闭溶液洗涤3次,每次5分钟后,用HRP缀合的山羊抗兔二抗(在BSA封闭溶液中1:1000稀释)孵育膜,在室温下温和搅拌1小时。
    11. 使用ECL检测试剂(根据制造商的方案)和Alliance 4.7软件进行Western印迹图像采集。
      注意:在与对照IgG裂解物特异性结合的情况下,应进行预清除步骤。向每个样品中加入100μl(50%珠/缓冲液)Protein G Sepharose珠,并在4℃下轻轻旋转孵育1小时。旋转珠子和收集上清液。按照步骤C3中所述加入免疫沉淀抗体。

数据分析

通过使用Alliance 4.7软件(UVITECH)对获得的图像进行密度测定分析来确定免疫沉淀的靶蛋白的定量。使用UVIband成像仪软件(Amersham)测定对应于Input(总裂解物的1/20)和免疫沉淀级分的条带的体积。免疫沉淀的量相对于相应的输入标准化。

代表数据

代表性实验的结果如图1所示。


图1.在HEK-293细胞中异位表达或在小鼠脑中内源性存在的PSD-95的MPM-2介导的免疫沉淀。在HEK-293细胞中异位表达的FLAG-PSD-95被免疫沉淀与抗MPM-2抗体。如所示,每个样品运行两次以用抗FLAG和抗MPM2抗体进行免疫印迹。小鼠IgG用作阴性对照。 B.PSD-95从脑提取物中沉淀,并使用抗MPM2抗体在Western印迹中揭示。星号表示由于二抗与免疫沉淀中使用的IgG重链的交叉反应性导致的背景条带 注意:只有一部分PSD-95在S-T/P共有基序处磷酸化(比较输入泳道)。

食谱

  1. 磷酸盐缓冲盐水(1x PBS)
    137 mM NaCl 2.7 mM KCl
    4.3mM Na 2 HPO 4
    1.47mM KH 2 PO 4 sub/
    调节至最终pH为7.4
    注意:在PBS-T中加入0.1%吐温20。
  2. MPM-2裂解缓冲液
    50mM Tris-HCl(pH7.5) 1%NP-40
    0.5%Triton X-100 150mM NaCl 10%甘油 10 mM原钒酸钠 1mM的氟化钠 1%磷酸酶抑制剂混合物1
    1x cOmplete TM 无EDTA蛋白酶抑制剂混合物
  3. 磷酸酶抑制剂
    1mM原钒酸钠 1mM的氟化钠 磷酸酶抑制剂鸡尾酒1
  4. IP洗涤缓冲液
    50mM Tris-HCl(pH7.5) 100 mM NaCl
    0.1%Tween 20
    0.1%SDS
    10 mM原钒酸钠 1mM的氟化钠 1%磷酸酶抑制剂混合物1
    1x cOmplete TM 无EDTA蛋白酶抑制剂混合物
  5. 2x Laemmli样品缓冲液
    100 mM Tris-HCl(pH 6.8)
    4%SDS
    20%甘油 1%2-巯基乙醇 0.001%溴酚蓝
  6. Western印迹运行缓冲液
    25 mM Tris
    190 mM甘氨酸 0.1%SDS
  7. Western印迹转移缓冲液
    25mM Tris(pH8.3) 192 mM甘氨酸 10%甲醇
    0.1%SDS
  8. Tris缓冲盐水-Tween 20(1×TBS-T) 50mM Tris(pH7.6) 150mM NaCl 0.1%Tween 20
  9. BSA封闭溶液(也用作抗体孵育缓冲液)
    1x TBS-T
    2.5%BSA

注意:所有缓冲液都储存在RT,除了BSA封闭溶液保持在4℃(不超过一周)。样品缓冲液不用2-巯基乙醇储存,在使用前新加入。只有免疫沉淀过程所需的缓冲液体积在冰上预冷却,并在即将使用前提供蛋白酶和磷酸酶抑制剂。将所有抑制剂重悬浮于灭菌的MilliQ水中,等分并保存在-20℃。原钒酸钠制备为在无菌MilliQ水中的200mM储备溶液,将pH调节至10.0。在该pH下,溶液是黄色的。为了确保钒酸盐单体的存在,将溶液煮沸直至半透明,并将pH再调节至10.0。

致谢

我们感谢S. Vicini博士(乔治城大学医学院,华盛顿特区,美国),提供PSD-95 cDNA。这项工作得到了Beneficentia基金会(BEN 2014/08)和Telethon(GGP11043)的资助。

参考文献

  1. Antonelli,R.,De Filippo,R.,Middei,S.,Stancheva,S.,Pastore,B.,Ammassari-Teule,M.,Barberis,A.,Cherubini,E.and Zacchi, 。  Pin1通过脯氨酰基修饰NMDA受体的突触内容, PSD-95的异构化。 J Neurosci 36(20):5437-5447。
  2. Chen,X.,Vinade,L.,Leapman,RD,Petersen,JD,Nakagawa,T.,Phillips,TM,Sheng,M。和Reese,TS(2005)。  质量的突触后密度和三个关键分子的枚举。 Proc Natl Acad Sci USA 102(32):11551-11556。
  3. Craven,SE,El-Husseini,AE和Bredt,DS(1999)。  突触靶向由脂质和蛋白质基序介导的突触后密度蛋白PSD-95。 Neuron 22(3):497-509。
  4. Davis,FM,Tsao,TY,Fowler,SK和Rao,PN(1983)。  有丝分裂细胞的单克隆抗体。美国国家科学院美国 80(10):2926-2930。
  5. Lu,KP(2004)。  固定单元格信号,癌症和阿尔茨海默氏病。


    趋势生物化学 29(4):200-209。
  6. Lu,KP,Hanes,SD和Hunter,T。(1996)。  调节有丝分裂所必需的人类肽基脯氨酰异构酶。 Nature 380(6574):544-547。
  7. Lu,KP,Liou,YC和Zhou,XZ(2002)。  固定脯氨酸定向的磷酸化信号传导。 Trends Cell Biol 12(4):164-172。
  8. Lu,KP和Zhou,XZ(2007)。  脯氨酰异构酶PIN1:磷酸化信号传导和疾病中的关键新扭曲。 Nat Rev Mol Cell Biol 8(11):904-916。
  9. Wulf,G.,Finn,G.,Suizu,F。和Lu,KP(2005)。  磷酸化特异性脯氨酰异构化:是否有基础主题? Nat Cell Biol 7(5):435-441。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Antonelli, R. and Zacchi, P. (2016). MPM-2 Mediated Immunoprecipitation of Proteins Undergoing Proline-directed Phosphorylation. Bio-protocol 6(23): e2046. DOI: 10.21769/BioProtoc.2046.
  2. Garré, J. M., Yang, G., Bukauskas, F. F. and Bennett, M. V. (2016). FGF-1 triggers Pannexin-1 hemichannel opening in spinal astrocytes of rodents and promotes inflammatory responses in acute spinal cord slices. J Neurosci 36(17): 4785-4801.
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