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Sep 2018

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Combining Gel Retardation and Footprinting to Determine Protein-DNA Interactions of Specific and/or Less Stable Complexes
结合凝胶阻滞和足印法测定特异性和/或不稳定复合物的蛋白质-DNA相互作用   

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Abstract

DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to interpret if the binding of the protein to the DNA is weak, the protein has a fast off-rate, or if several different protein-DNA complexes are formed. Our protocol differs from traditional footprinting protocols, because it provides a method to isolate the protein-DNA complex from a native gel after treatment with the footprinting agent, thus removing the bound DNA from the free DNA or other protein-DNA complexes. The DNA is then extracted from the isolated complex before electrophoresis on a sequencing gel to determine the footprinting pattern. This analysis provides a possible solution for those who have been unable to use traditional footprinting methods to determine protein-DNA contacts.

Keywords: DNase I footprinting (DNasel足印分析), KMnO4 footprinting (KMnO4足印分析), Gel retardation assays (凝胶阻滞分析), EMSAs (EMSAs), Protein-DNA interactions (蛋白质-DNA相互作用)

Background

Nuclease/chemical footprinting is a classic method to probe protein-DNA interactions (Galas and Schmitz, 1978; Sasse-Dwight and Gralla, 1989; Hampshire et al., 2007). In this technique, the binding of a protein to a particular region of DNA inhibits (protection) or increases (enhancement) the ability of the nuclease or chemical to cleave the DNA. Consequently, if using 5’-end labeled DNA, a specific pattern of protection/enhancement will be revealed by incubation of the protein-DNA complex with the cleaving reagent and the subsequent electrophoresis of the DNA on a sequencing gel. However, in order to obtain a clear, interpretable pattern, the protein must bind the DNA relatively tightly during the cleavage process. If the protein(s) of interest binds to the DNA weakly and/or if the protein has a fast off-rate, traditional footprinting methods are likely to fail. Furthermore, sometimes multiple complexes can be formed with differing protein-DNA contacts. In this case, the footprint pattern will only reveal the full ensemble of interactions rather than those associated with a discrete complex.

Here, we have developed a straightforward method to treat protein-radiolabeled DNA complexes with DNase I or KMnO4 followed by isolation of the bound complex using an Electrophoretic Mobility Shift Assay (EMSA). In this assay, the DNA is incubated with the protein(s) of interest, and free DNA is separated from protein-bound DNA by electrophoresis on a native gel. This separation occurs because the protein-DNA complex migrates more slowly than the free DNA on the gel. The protein-DNA complex of interest is then extracted from the excised polyacrylamide gel slice, and the DNA is isolated and electrophoresed on a sequencing gel to determine the footprint. In many instances, this protocol allows users to obtain clear footprint patterns even when investigating less stable and/or weakly bound complexes. For DNase I reactions, the protection pattern with or without protein(s) indicates protein binding site(s) while the hypersensitivity site(s)/enhancement pattern represents DNA bending, kinking, and/or looping. For KMnO4 reactions, enhancement bands represent regions containing single-stranded thymine residues, such as within an open transcription bubble. However, the procedure could be adapted to other types of enzymatic or chemical reagents, which would yield different types of patterns.

In the lab, we have successfully used this protocol to obtain clear footprints of protein-DNA complexes for transcriptional activators in two different bacterial systems: Bordetella pertussis BvgA (Boulanger et al., 2015) and Vibrio cholerae VpsR (Hsieh et al., 2018 and 2020) in the presence and absence of RNA polymerase (RNAP). In the case of VpsR, we separated the protein-DNA complexes formed in the presence or absence of the small molecule cyclic-di-GMP (c-di-GMP) from the free DNA (Figure 1A, 1B) in order to obtain direct patterns of the complexes themselves (Figure 1C). In the case of phosphorylated response regulator BvgA (BvgA~P), while only one complex, the open complex (RPo), was formed by RNAP in the absence of ribonucleoside triphosphates (rNTPs) (Figure 2A, lanes 1 vs. 2), two complexes, both RPo and the initiating complex (RPi), were formed by RNAP in the presence of rNTPs (Figure 2A, lane 3). Consequently, we used this technique to separate RPi from the free DNA and from RPo, allowing us to visualize the DNase I footprint of each complex separately (Figure 2B, lanes 2 and 3). We also employed this method together with KMnO4 footprinting to determine the position of the transcription bubble within RPo formed by RNAP with non-phosphorylated BvgA (Figure 2A, lane 4) or within RPo formed by RNAP and BvgA~P (Figure 2A, lane 2). This yielded the KMnO4 footprints seen in Figure 2C, lanes 1 and 2, respectively. We were then able to compare these results to those obtained without the complex isolation step (Figure 2D, lanes 1 and 2), allowing us to determine how the stable complexes differed from the mixture of complexes that were formed in solution.


Figure 1. Example of EMSA/DNase I footprinting. Figure has been adapted and reprinted with permission from Hsieh et al., 2018. (A) and (B) EMSA gels showing complexes formed by the V. cholerae regulator VpsR bound to the promoter DNA for the V. cholerae gene vpsL (PvpsL) in the presence and absence of the small molecule c-di-GMP and in the absence (A) and presence (B) of RNA polymerase (RNAP), as indicated. The position of the free DNA is indicated by grey arrows while shifted complexes are represented by black arrows. Binding reactions in (A) contained 5 nM 32P-labeled nontemplate PvpsL harboring -97 to +103 relative to the transcription start site, 1 μg poly (dI-dC) as competitor, and increasing amounts of VpsR from 0 to 2 μM in the absence (lanes 1-5) and presence (lanes 6-10) of 50 μM c-di-GMP. Binding reactions in (B) contained 0.04 μM PvpsL, 0.14 μM reconstituted RNAP (σ:core ratio of 2.5:1), 1.4 μM VpsR, and 50 μM c-di-GMP, as indicated, and the reactions were competed with 500 ng heparin for 15 s. (C) Sequencing gels showing the DNase I products from isolated complexes. In these cases, reactions were incubated with 0.3 U of DNase I at 37 °C for 30 s before loading on the EMSA gel, the free DNA and complexes were excised and isolated, and after extraction, the DNA was electrophoresed on the gel. GA indicates G+A ladder. A schematic of the -10 element, -35 element, and the +1 position of PvpsL is shown to the right of the image. Protection pattern and hypersensitivity sites are indicated by black rectangular boxes and thin black arrows, respectively. Dashed red box represents the VpsR binding site.


Figure 2. Example of EMSA/DNase I footprinting and of KMnO4 footprinting with and without isolation of protein-DNA complexes from EMSA gel. Figure has been adapted and reprinted with permission from Boulanger et al., 2015. (A) EMSA gel showing complexes formed by the B. pertussis response regulator BvgA either phosphorylated (BvgA~P) or non-phosphorylated (BvgA), the promoter for the B. pertussis gene fim3 (Pfim3), and RNAP. Binding reactions contained 0.05 pmol DNA, 5 pmol BvgA or BvgA~P, and 0.75 pmol reconstituted RNAP (σ:core ratio of 2.5:1). All samples were competed with 200 μg/ml heparin and as indicated, rNTPs (GTP, ATP, and CTP) were added. Free DNA is represented by a grey arrow while the open complex (RPO) is labeled with a black arrow and the initiating complex (RPI) is represented by a dashed black arrow. (B) Sequencing gels showing DNase I footprints at Pfim3 obtained from binding reactions with 0.36 U of DNase I and the subsequent EMSA isolation step. A schematic of the binding region as well as -35 , -10, and +1 is to the right of the image. Hypersensitive sites at -15 and -80, observed with RPi, but not with RPo, are indicated with the ‘*’. (C) and (D) Sequencing gels showing KMnO4 footprints in the region of position +1 of Pfim3 obtained by treating the binding reactions with 2.5 mM KMnO4 for 2.5 min at 37 °C and then either obtaining complexes from an EMSA gel and isolating the DNA before running the sequencing gel (C) or using the binding reaction products directly without the EMSA isolation step (D). Binding reactions contained 0.5 pmol DNA, 12 pmol BvgA or BvgA~P and 1.125 pmol reconstituted RNAP (σ:core ratio of 2.5:1). GA represents G+A ladder.

Materials and Reagents

Note: All reagents are stored at room temperature unless otherwise indicated.

  1. UltraCruz® Autoradiography Tape (Santa Cruz Biotechnology, catalog number: sc-200214)

  2. Clear plastic wrap, such as Saran Wrap or Glad Cling Wrap

  3. Razor blade (Personna Gem, catalog number: 62-0179 or equivalent)

  4. 1.7 ml microtubes (sterile, RNase- and DNase-free) (Genesee Scientific, catalog number: 24-282S)

  5. 0.2 ml PCR tubes (Axygen, catalog number: PCR-02-C)

  6. 1.5 ml Pestle (Fisher Scientific, catalog number: 12-141-364)

  7. 50 ml, 0.22 micron Millipore Steriflip filtration system (Sigma-Aldrich, catalog number: SCGP00525)

  8. Ultrafree MC Centrifugal Filters (Millipore, catalog number: UFC30HV00)

  9. 8” x 10” Hyblot CL Autoradiography Film (Denville Scientific, catalog number: E3018)

  10. 14” x 17” Hyblot CL Autoradiography Film (Denville Scientific, catalog number: E3031)

  11. TranScreen-HE (Kodak, catalog number: 881-1457)

  12. 8” x 10” Film cassettes (Research Products International, catalog number: 420180)

  13. 14” x 17” Film cassettes (Research Products International, catalog number: 421417)

  14. 8” x 10” Film cassette security bag (Thomas Scientific, catalog number: E3753-1)

  15. 14” x 17” Film cassette security bag (Thomas Scientific, catalog number: E3753-0)

  16. Purified proteins to be tested and stored at the appropriate temperature for the particular protein

  17. Oligonucleotides that anneal to the 5’-end of the DNA regions (usually 20-30 basepairs (bp)) and are used in PCR to amplify the DNA region needed for footprinting, dissolved in ddH2O at 50 pmol/μl and stored at -20 °C (We recommend using PCR to generate a DNA fragment of 200 to 300 bp). We have obtained oligonucleotides from Integrated DNA Technologies. However, oligonucleotides can be purchased from many sources.

  18. Genomic DNA or plasmid DNA containing the protein-binding DNA region that can be used as a template for PCR amplification, dissolved in ddH2O or TE (see below) at 100 ng/ml and stored at 4 °C (genomic DNA) or at -20 °C (plasmid DNA)

  19. [γ-32P]-ATP (3,000 Ci/mmol, 10 mCi/ml, 250 μCi) (PerkinElmer, catalog number: BLU002A; stored at -20 °C)

  20. Optikinase (10 U/μl) and 10x Optikinase buffer (USB Affymetrix, catalog number: 78334Y; stored at -20 °C)

  21. Pfu Turbo polymerase (2.5 U/μl) and 10x Pfu buffer (Agilent Technologies, catalog number: 600252; stored at -20 °C)

  22. dNTP mix (New England Biolabs, catalog number: N0447, 10 mM; stored at -20 °C)

  23. DNase I (2 U/μl) and 10x DNase I buffer (Life Technologies, catalog number: AM2222; stored at -2 °C)

  24. Heparin (Sigma-Aldrich, catalog number: H3149, 500 μg/ml dissolved in ddH2O; stored at -20 °C)

  25. Poly(deoxyinosinic-deoxycytidylic) (Poly(dI-dC)) acid sodium salt (Sigma-Aldrich, catalog number: P4929, 1 mg/ml dissolved in ddH2O; stored at -20 °C)

  26. Urea Ultrapure (Invitrogen, catalog number: 15505-050)

  27. Petroleum jelly

  28. Large binder clips

  29. 40% 19:1 Acrylamide:bis solution (Bio-Rad Laboratories, catalog number: 1610144; stored at 4 °C)

  30. 40% 37.5:1 Acrylamide:bis solution (Bio-Rad Laboratories, catalog number: 1610148; stored at 4 °C)

  31. Tetramethylethylenediamine (TEMED) (Bio-Rad Laboratories, catalog number: 1610800; stored at 4 °C)

  32. TE-saturated Phenol, pH 8.0 (Sigma-Aldrich, catalog number: 77607; stored at 4 °C)

  33. Phenol:chloroform:isoamyl alcohol (25:24:1 v/v, pH 7.9) (Life Technologies, catalog number: AM9732; stored at 4 °C)

  34. 190 proof ethanol (Warner-Graham Company; stored at -20 °C)

  35. Drawn-out plastic pipet (Fisher Scientific, catalog number: 13-711-27)

  36. GlycoBlueTM Coprecipitant (Invitrogen, catalog number: AM9515; stored at -20 °C)

  37. 10x TBE Buffer (Quality Biological, catalog number: 351-001-131)

  38. 50x TAE Buffer (Quality Biological, catalog number: 351-008-131)

  39. TE pH 8.0 (Quality Biological, catalog number: 351-011-131)

  40. 0.5 M EDTA, pH 8.0 (Quality Biological, catalog number: 351-027-101)

  41. Formamide (Sigma-Aldrich, catalog number: 47670)

  42. 1-Butanol (Fisher Scientific, catalog number: A399-500)

  43. 3 M Sodium Acetate (Sigma-Aldrich, catalog number: 71196)

  44. Dry ice

  45. 6x Dye Loading solution for native gel (Fermentas, catalog number: R0611)

  46. Xylene cyanol (XC) dye (Sigma-Aldrich, catalog number: X4126)

  47. Bromophenol blue (BPB) dye (Sigma-Aldrich, catalog number: B0126)

  48. AG® 501-X8(D) mixed bed resin (Bio-Rad Laboratories, catalog number: 1426425)

  49. Salmon sperm DNA (Sigma-Aldrich, catalog number: D1626; 10 mg/ml, 5 mg/ml, and 1 mg/ml dissolved in ddH2O; stored at -20 °C)

  50. ddH2O

  51. APS (Bio-Rad Laboratories, catalog number: 1610700)

  52. Ammonium acetate (Sigma-Aldrich, catalog number: A7330)

  53. Calcium chloride dihydrate (Sigma-Aldrich, catalog number: C3306)

  54. KMnO4 (Sigma-Aldrich, catalog number: 223468)

  55. Magnesium acetate tetrahydrate (Sigma-Aldrich, catalog number: M5661)

  56. Formic acid stock (88%) (Sigma-Aldrich, catalog number: 399388). 4% diluted in ddH2O (see Recipes)

  57. 50 mM KMnO4 (Potassium permanganate) (see Recipes)

  58. 10% Ammonium persulfate (APS) (see Recipes)

  59. 10 M Ammonium acetate (see Recipes)

  60. 1 M Magnesium acetate (see Recipes)

  61. 20% SDS (Quality Biological, catalog number: 351-066-721); 1% SDS diluted in ddH2O (see Recipes)

  62. 2 mM CaCl2 (Calcium chloride) (see Recipes)

  63. Piperidine stock (10 M) (Sigma-Aldrich, catalog number: 104094); 2 M and 100 mM diluted in ddH2O (see Recipes)

  64. Formamide loading dye; stored at -20 °C (see Recipes)

  65. 2-mercaptoethanol stock (14.3 M) (Sigma-Aldrich, catalog number: M3148). 1 M diluted in ddH2O (see Recipes)

  66. 70% ethanol (see Recipes; stored at -20 °C)

  67. Diffusion buffer (see Recipes)

Equipment

  1. Suitable space for working with 32P radioactivity

  2. Geiger counter to monitor radioactivity and contamination

  3. Plexiglass shield to protect user from radioactivity

  4. Plexiglass box for 32P waste

  5. 150 ml, 0.22 micron filter apparatus (Nalgene, catalog number: 125-0020 or equivalent)

  6. PCR Machine

  7. Vortex (such as Vortex Genie 2, Scientific Industries)

  8. Heating block that that can warm up to 95 °C

  9. Vertical gel box - medium size (~7”W x ~9”H) (such as Cole-Parker Vertical Single Adjustable Slab Gel System, catalog number: EW-28570-00 or HoeferTM Air-cooled Vertical Electrophoresis Unit, catalog number: SE400)

  10. Vertical gel box- large size (~14”W x ~17”H) (such as LABRepCo Model S2 Sequencing gel Electrophoresis Apparatus, catalog number: 21105036 or BTLab Systems Nucleic Acid Sequencing Electrophoresis cell (330 x 420 mm), catalog number: BT210)

  11. Elutrap electroelution system (GE Healthcare, catalog number: 10447711)

  12. Power supply box for electrophoresis (Bio-Rad Laboratories)

  13. Small Microcentrifuge, such as Benchmark MyFuge Mini that spins at 6,000 rpm/2,000 x g

  14. Benchtop Microcentrifuge, such as Eppendorf Centrifuge 5425 that can spin at ≥ 14,000 x g (Eppendorf, catalog number: 5405000646)

  15. Speed Vacuum (we use Thermo Electron Corp. Savant DNA 120 SpeedVac System, whose speed is 1600 rpm; Thermo Fisher Scientific, catalog number: 13442549)

  16. Scintillation Counter

  17. Film Developer (or phosphoimager)

  18. Densitometer to scan autoradiographs [we use a GS-800 Calibrated Imaging Densitometer from Bio-Rad Laboratories, catalog number: 170-7983 (discontinued); new model is GS-900, catalog number: 170-7989]

  19. Glass plates with spacers and combs to fit medium sized gel apparatus (Gel Company; we suggest a gel thickness of 1 mm)

  20. Glass plates with spacers and combs to fit large sized gel apparatus (Gel Company; we suggest a gel thickness of 1 mm)

Software

  1. Software to operate densitometer (We use Quantity One from Bio-Rad Laboratories, catalog number: 1709601)

Procedure

Unless otherwise indicated, all reactions are assembled on ice, and centrifugations are performed at room temperature.

  When working with 32P, use safety protocols for handling radioactivity, checking surfaces for contamination, and disposal of waste. We recommend using RNase/DNase-free, sterile ddH2O for solutions and appropriate precautions to minimize the possibility of DNase contamination.

  1. Make 5′-32P DNA end-labeled on either the nontemplate or template strand

    1. Kinase reaction: 5’-32P end-label either template or nontemplate strand oligonucleotide.

      In the 0.2 ml PCR tube combine the following:

      7 μl [γ-32P]-ATP

      1 μl Optikinase enzyme

      1 μl 10x Optikinase buffer

      1 μl oligonucleotide (50 pmol/μl)

      Incubate at 37 °C for 30 min. Inactivate enzyme by incubating at 65 °C for 10 min.

    2. Assemble PCR reaction.

      Add the following to the kinase reaction:

      76 μl ddH2O

      10 μl 10x Pfu buffer

      1 μl 10 mM dNTP mix

      1 μl plasmid DNA or gDNA (100 ng/ml)

      1 μl other (nonlabeled) oligonucleotide (50 pmol/μl)

      1 μl Pfu Turbo enzyme (2.5 U/μl).

    3. Perform PCR to generate labeled DNA fragment with the following cycles:

      1. 95 °C for 2 min to denature the DNA

      2. 95 °C for 30 s

      3. Temperature of (m-5 °C)* for 30 s

      4. 72 °C for 30 s

      5. Repetition of steps b-d for 25-35 cycles

      6. 72 °C for 10 min

      7. 4 °C for storage.

      * m-5 °C–temperature that is 5 °C lower than the lowest primer melting temperature.

    4. Prepare 4% polyacrylamide gel for the medium-sized gel apparatus the day before to ensure complete gel solidification and improved resolution.

      Assemble glass plates using petroleum jelly to seal at corners of side and bottom spacers and large binder clips to keep plates together.

      Mix together:

      4 ml 40% acrylamide:bis 19:1

      34.8 ml ddH2O

      0.8 ml 50x TAE

      0.4 ml 10% APS

      Filter solution through a 150 ml, 0.22 micron filter apparatus. Add 12 μl TEMED to the filtrate, swirl gently to mix (avoid introducing air bubbles), and immediately pour gel and insert comb. Carefully cover comb area with plastic wrap. Use binder clips over the plastic wrap at the top of gel to make a tight seal between the glass plates and the comb. Allow gel to solidify overnight at room temperature.

    5. Electrophorese PCR-generated labeled DNA fragment on the 4% vertical polyacrylamide gel.

      Place the gel into the medium-sized gel apparatus. After removing comb, mark the well positions on the front of the glass plates with a permanent marker. (This significantly improves slot visualizing during loading.) Pre-run gel at 100 V in 1x TAE for 1 h. Add 6x Dye Loading solution to the PCR sample volume. Load all of the sample on the gel. Run at 140 V in 1x TAE until the BPB component of the 6x loading dye reaches ~13 cm from the origin (~2 h).

    6. Excise labeled DNA from gel.

      Dismantle gel. Remove top glass plate. Wrap bottom plate/gel with plastic wrap. Mark corners of plastic-wrapped gel with autoradiography tape. Follow instructions for marking the tape. Expose gel to film for a few min. (Approximately 2-3 min should be sufficient.) Develop film and determine the position of the radiolabeled DNA on the gel by aligning film to the gel that is still wrapped. Make a stencil by using a razor blade to cut out desired labeled DNA band(s) on the developed film. Align stencil to plastic-wrapped gel. Using the stencil, mark the position of the labeled DNA on the plastic-wrapped gel with a permanent marker. Excise band with a razor blade. Use a clean razor for each sample. Be sure to remove the plastic wrap once labeled DNA is excised from gel. Note orientation of the gel as you remove the slice (- vs + end).

    7. Isolate the labeled DNA by electroelution.

      Assemble Elutrap per instruction manual. Fill chambers with X TAE. Load excised labeled DNA in the same orientation as it was running in the gel. (Be sure the ‘–‘ end of gel is towards the ‘–‘ end of the Elutrap and the ‘+‘ end of the gel is towards the ‘+‘ end of the Elutrap.) Electrophorese at 200 V for 1 h. Collect solution containing the labeled DNA and place it into a 1.7 ml microtube. Repeat electrophoresis, collect solution, and add it to the same tube.

    8. Precipitate labeled DNA.

      Evaporate labeled DNA solution to approximately 200 μl in the speed vacuum. (Note: This can take up to an hour or longer; use the ambient temperature setting and do NOT turn on heat to expedite drying). Add 200 μl phenol (TE-saturated). Mix 30 s, centrifuge at 2,000 x g for 1 min in small microcentrifuge and transfer the labeled DNA in the top (aqueous) layer to a clean microtube. Add 5x volume of 190 proof ethanol and volume of 10 M ammonium acetate to the phenol-extracted, aqueous layer containing the labeled DNA, mix well, and incubate on dry ice for 1 h or at -20 °C overnight. Centrifuge in benchtop microcentrifuge at highest speed (≥14,000 x g) for 30 min, remove ethanol solution with drawn-out pipet, and wash precipitated labeled DNA pellet with 100 μl ice cold 70% ethanol. Centrifuge as above for 5 min, remove 70% ethanol, and dry the labeled DNA in the speed vacuum for 2-3 min at room temperature. Resuspend pellet in 20 μl TE. This assumes a 10% to 40% incorporation of 32P into the DNA and a yield of > 25%. However, if desired, the incorporation rate and yield can be quantified by using TCA (trichloroacetic acid) precipitation (see here).


  2. Prepare a G+A sequencing ladder (Maxam and Gilbert,1977)

    1. In a 1.7 ml microtube, combine the following:

      1 μl labeled DNA

      1 μl 1 mg/ml salmon sperm DNA

      8 μl TE

    2. Add 1 μl 4% formic acid (see Recipes).

    3. Incubate at 37 °C for 45 min.

    4. Place tube on ice and add 150 μl 2 M piperidine (see Recipes).

    5. Incubate at 90 °C for 30 min.

    6. Put tube on ice, and add 5 μl 10 mg/ml salmon sperm DNA.

    7. Add 1 ml 1-butanol, vortex to mix thoroughly, and then centrifuge in the benchtop microcentrifuge at ≥ 14,000 x g for 5 min. Carefully remove and discard supernatant.

      Note: Pellets formed by butanol precipitation have a tendency to float away from the side of the tube so it is important to make sure that the pellet is not discarded when removing the supernatant.

    8. Add 150 μl of 1% SDS to the pellet (see Recipes).

    9. Add 1 ml butanol, vortex to mix thoroughly, and then centrifuge in the benchtop microcentrifuge at ≥ 14,000 x g for 5 min. Carefully remove and discard supernatant.

    10. Wash pellet two times with 0.5 ml butanol, centrifuging for 1 min in the benchtop microcentrifuge at ≥ 14,000 x g after each wash. Carefully remove and discard supernatant.

    11. Dry pellet in the speed-vac for 1-2 min at room temperature.

    12. Add 10 μl formamide load solution to dissolve and store at -20 °C.


  3. Perform DNase I or KMnO4 reaction

    Note: The amount of protein and the specific binding buffer will be dependent on the protein being used. As a starting point, use conditions in which you know that your protein is active or see conditions listed in Boulanger et al. (2015) and Hsieh et al. (2018 and 2020).

    1. DNase I reactions

      In a total 10 μl volume, incubate protein of interest (or just the protein’s buffer as the negative control), the labeled DNA, and binding buffer appropriate for the protein to bind to the DNA. Additionally, the buffer should contain 2 mM CaCl2 for the DNase I activity. The temperature should be appropriate for the protein being tested, but typically will range between room temperature and 37°C. We recommend using 0.1 to 0.5 pmol of DNA per reaction. The ratio of protein:DNA can be altered depending on what conditions are being tested. For good binding, we recommend starting with at least a 5 to 10 fold excess of protein to the labeled DNA. To eliminate unstable and non-specific complexes, we also recommend the addition of 1 μl of 1 mg/ml poly (dI-dC) in the binding reaction or 0.5 to 1 μl of 500 μg/ml heparin after the binding reaction is completed. Dilute the stock DNase I enzyme (2 U/μl) in 1x DNase I buffer to the desired concentration. We recommend trying a range of DNase I concentrations. We have previously used ~0.3 U total. To initiate the DNase I reaction, add 1 μl diluted DNase I enzyme (or 1 μl 1x DNase I buffer as the control). Final reaction volume is 11 μl. Reactions can also be scaled up. Mix reaction components by tapping finger on tube 2-3 times and quickly centrifuge the sample using the small microcentrifuge at 2,000 x g. This step should take only ~15 s. Place sample in 37°C heating block, and incubate for 30 s. (This time can be varied if more or less cleavage is desired.) Immediately load the sample on gel that is already running at 100 V/h. See below: Step D3.

    2. KMnO4 reactions

      Incubate protein of interest (or just the protein’s buffer as the negative control) with the labeled DNA in an appropriate binding buffer (10 μl total volume) at 37°C to initiate formation of single-stranded regions of the DNA, such as in an open transcription complex when using RNAP. We recommend using 0.1 to 0.5 pmol of labeled DNA. After this incubation, a competitor, such as heparin (1 μl of 500 μg/ml solution), can be added with an additional incubation of ~1 min to remove unstable complexes. Add 0.5 μl of 50 mM KMnO4 (see Recipes) (or 0.5 μl of ddH2O as the negative control). Be sure to make the KMnO4 solution on the day of use. Incubate for 2.5 min at 37°C. Quench the reaction by adding 5 μl 1 M 2-mercaptoethanol (see Recipes). Immediately load sample onto gel that is already running at 100 V/h. See below: Step D3.


  4. Electrophorese DNase I-treated or KMnO4-treated complexes on a 4% acrylamide native gel

    1. Prepare 4% polyacrylamide gel for the large-sized gel apparatus the day before to ensure complete gel solidification and improved resolution.

      Assemble glass plates using petroleum jelly to seal at corners of spacers and large binder clips to keep plates together.

      Mix together:

      12 ml 40% acrylamide:bis 37.5:1

      12 ml 10x TBE

      95.28 ml ddH2O

      0.72 ml 10% APS.

      Filter solution through a 150 ml 0.22 micron filter unit. Remove 4 ml of gel solution, add 4 μl TEMED to this aliquot, mix well, and pour into plates. Once this portion of the gel solidifies as a plug (~15 min), add 20 μl TEMED to remaining gel solution, mix well by inverting, but do not introduce air bubbles. Immediately pour into plates. Remove any air bubbles by tapping on glass plates. Lay horizontally on a support and immediately insert comb. Carefully cover comb area with plastic wrap. Use binder clips over the plastic wrap at the top of gel to make a tight seal between the glass plates and the comb. Allow gel to solidify overnight at room temperature.

    2. Pre-run gel at 100 V/h for 2 h. (Be sure to mark the well positions on the front of the glass plates with a permanent marker. This significantly improves slot visualizing during loading.)

    3. While gel is running at 100 V/h, load all of sample for one reaction in one lane. To ensure good separation, skip one lane when loading each new sample. (If more sample will be needed, the same reaction can be performed multiple times and loaded into multiple lanes. The products are then combined after excision of the gel slices.)

    4. Run the gel at 380 V for 3 h.

    5. Dismantle gel. Remove top plate and wrap gel/bottom plate with plastic wrap.


  5. Isolate and extract labeled DNA from 4% acrylamide native gel

    1. Using autoradiography tape, mark each corner of the plastic-wrapped gel. This will help with stencil alignment.

    2. Place unexposed film on gel in a film cassette in the dark room.

    3. Expose film overnight.

    4. Develop film.

    5. As detailed in Step A6 make a stencil, but in this case, cut out both the free labeled DNA band and any desired complexes from the film. Take the minimum amount of gel needed to obtain all of the radioactivity. Slices are typically about 4-5 mm wide (the width of a gel lane) and 3 mm long. However, longer slices can be taken if necessary. Be sure to label.

    6. Place each gel slice in an empty 1.7 ml microtube.

    7. Crush the polyacrylamide gel with a 1.5 ml pestle in the microtube.

    8. Add 200 μl diffusion buffer (see Recipes) per sliced band. (If multiple slices were combined, add the appropriate amount of diffusion buffer.)

    9. Incubate tubes at 60 °C for at least 2 h or at room temperature overnight.

    10. Centrifuge in the benchtop microcentrifuge at ≥ 14,000 x g for 5 min.

    11. Decant or carefully remove solution with pipet tip.

    12. Transfer solution to a MC centrifugal filter.

    13. Centrifuge the MC centrifugal filter in benchtop microcentrifuge at ≥14,000 x g for 5 min to remove traces of polyacrylamide.

    14. Transfer flow-through to a clean 1.7 ml microtube.

    15. In a speed vacuum, reduce volume to ~200 μl for DNase I reactions and ~100 µl for KMnO4 reactions, if necessary.

      Note: This can take up to an hour or longer; use the ambient temperature setting and do NOT turn on heat to expedite drying.

    16. For DNase I reactions, phenol-extract solution. Add 200 μl phenol:chloroform:isoamyl alcohol, mix 30 s, centrifuge in small microcentrifuge at 2,000 x g for 1 min, and transfer the top (aqueous) layer containing the labeled DNA to a clean 1.7 ml microtube.

      Ethanol precipitate DNA. Add 5x volume of 190 proof ethanol, DNA volume of 10 M ammonium acetate (see Recipes), and 1 μl GlycoBlue to the phenol-extracted aqueous DNA layer, mix well, incubate on dry ice for 1 h or at -20 °C overnight, centrifuge in benchtop microcentrifuge at ≥ 14,000 x g for 30 min, remove ethanol/ammonium acetate/GlycoBlue solution with drawn-out pipet, wash labeled DNA pellet with 100 μl ice cold 70% ethanol, centrifuge as above for 5 min, remove 70% ethanol, and dry in speed vacuum for 2-3 min at room temperature.

    17. For KMnO4 reactions, ethanol precipitate the labeled DNA. Add 10x volume of 190 proof ethanol, mix well, incubate at -20 °C for 20 min, centrifuge in benchtop microcentrifuge at ≥ 14,000 x g for 20 min, remove ethanol solution with drawn-out pipet, wash labeled DNA pellet with 100 μl ice cold 70% ethanol, centrifuge in benchtop microcentrifuge at ≥ 14,000 x g for 5 min, remove 70% ethanol, and dry in speed vacuum for 2-3 min. Resuspend pellet in 100 μl 100 mM piperidine (see Recipes) and incubate at 90 °C for 30 min to perform cleavage reaction. Place the sample on ice for 5 min prior to the addition of 2 μl of 5 mg/ml salmon sperm DNA. Add 1 ml 1-butanol, vortex sample to mix thoroughly, and centrifuge in benchtop microcentrifuge at ≥14,000 x g for 5 min (Note: Pellets formed by butanol precipitation have a tendency to float away from the side of the tube so it is important to make sure that the pellet is not discarded when removing the supernatant.). Wash with 10 μl 1-butanol, spin in benchtop microcentrifuge at ≥14,000 x g for 5 min and remove butanol wash. Dry samples in speed vacuum for 2-3 min. Resuspend pellet in 18.75 μl TE and 6.25 μl 3 M sodium acetate. Repeat ethanol precipitation as described at the beginning of step 17.

    18. Resuspend pellet in 10 μl formamide loading dye (see Recipes).

    19. Count samples in scintillation counter.


  6. Electrophorese labeled DNA and the G+A ladder on an 8% denaturing sequencing gel and image gel

    1. Prepare 8% denaturing, sequencing gel for the large-sized gel apparatus the day before to ensure complete gel solidification and improved resolution.

      Assemble glass plates using petroleum jelly to seal at corners of spacers and large binder clips to keep plates together.

      Combine in a 250 ml beaker:

      24 ml 40% acrylamide:bis 19:1

      53.3 g urea

      50.3 ml ddH2O

      1 g mixed bed resin

      Place beaker at 37°C until all of the urea has dissolved (This can require ≥20 min). The mixed bed resin will remain. Swirl to mix, and filter through a 150 ml 0.22 micron filter unit. Add 6 ml 10x TBE and 0.72 ml 10% APS to the filtrate. Pour a gel ‘plug’ by adding 4 μl TEMED to 4 ml of the above gel solution. Mix well and pour into glass plates. Wait to solidify (~15 min). Add 20 μl TEMED to remaining gel solution, mix well by inverting but do not introduce bubbles. Immediately pour gel. Remove air bubbles by tapping on glass plates. Lay horizontal on a support and immediately insert comb. Carefully cover comb area with plastic wrap. Use binder clips over the plastic wrap at the top of gel to make a tight seal between the glass plates and the comb. Allow gel to solidify overnight at room temperature.

    2. Pre-run gel at 500 V for ≥ 30 min, 750 V for ≥ 30 min, and then 1,000 V for ≥ 30 min in x TBE.

      Note: Be sure to mark the well positions on the front of the glass plates with a permanent marker. This significantly improves slot visualizing during loading.

    3. Stop pre-running gel. Heat samples at 95 °C for 2 min. During this time, rinse out each well with the running buffer using a drawn-out pipet to remove any urea that has diffused into the well.

    4. Load samples (as well as the G+A ladder lane to determine the sequence of the DNA). Load approximately the same cpm in each lane (based on the scintillation counter values). We recommend loading a minimum of 8,000 counts per lane. Lower amounts can be used, but will require extended film exposure times.

    5. Run gel at 1,000 V/h for ~3 h. (The exact time will depend on the region of the labeled DNA that needs to have the greatest resolution to observe the footprint.)

    6. Remove gel from apparatus. Remove the top plate and wrap the gel/bottom plate with plastic wrap.

    7. Place unexposed film on the plastic-wrapped gel/bottom plate in the dark room and store gel plate/gel/film in a film cassette placed in the cassette security bag in the -80 °C freezer. To help decrease static, you can also add an extra exposed film in between the unexposed film and the plastic-wrapped gel. Alternatively, a phosphor imaging screen and phosphor imager can be used instead.

    8. After desired amount of days, remove film from gel and develop. Add new film if additional exposures are needed. Consider using a TranScreen if the signal is weak, and signal amplification is needed.

    9. Scan autoradiograph on a densitometer. We use a GS-800 densitometer from Bio-Rad Laboratories.

Recipes

We recommend using RNase/DNase-free, sterile ddH2O for solutions to minimize the possibility of RNase or DNase contamination. Unless indicated otherwise, solutions are made and stored at room temperature.

  1. 4% formic acid

    9.1 μl of 88% stock plus 190.9 μl ddH2O

    Note: Make fresh right before use.

  2. 1% SDS

    7.5 μl of 20% SDS stock plus 142.5 μl ddH2O

  3. 10% APS

    1 g ammonium persulfate dissolved in 10 ml ddH2O

    Solution is freshly prepared before use.

  4. Formamide loading dye

    Mix 1 ml deionized formamide and 10 μl saturated solution of XC and 10 μl saturated solution BPB. Solution can be stored at -20°C for 1 week.

    Note: To deionize the formamide, add ~0.5 g of AG® 501-X8(D) mixed bed resin to 5 ml of formamide, incubate at room temperature for at least 10 min, and filter using a 0.22 micron Millipore Steriflip. To make saturated solution of either XC or BPB, add ~0.2 g of the dye to a sterile 1.7 ml tube and add 1 ml of ddH2O, vortex vigorously, and centrifuge briefly using the small microcentrifuge at 2,000 x g to pellet the undissolved powder. Remove 10 μl from the liquid above the remaining powder to add to the loading dye solution.

  5. 10 M Ammonium acetate

    Dissolve 77 g of ammonium acetate in 100 ml ddH2O and filter with 0.22 micron filter unit

  6. 2 mM CaCl2

    Dissolve 0.029 g calcium chloride dehydrate in 100 ml ddH2O and filter with 0.22 micron filter unit

  7. 50 mM KMnO4

    For the 250 mM stock, dissolve 3.95 g KMnO4 in 100 ml ddH2O

    Note: This can be stored at room temperature for several weeks.

    For 50 mM KMnO4, which should be made fresh on the day of use, mix 10 μl 250 mM KMnO4 with 40 μl ddH2O

  8. 100 mM piperidine

    Note: Solution is freshly prepared before use.

    10 μl of piperidine stock (10 M)

    990 μl ddH2O

  9. 2 M piperidine

    Note: Solution is freshly prepared before use.

    30 μl of piperidine stock (10 M)

    120 μl H2O

  10. 1 M 2-mercaptoethanol

    Note: Solution is stored at -20 °C and can be thawed/refrozen for months.

    7 μl of 2-mercaptoethanol stock (14.3 M)

    93 μl ddH2O

  11. 1 M Magnesium acetate

    Dissolve 21.45 g magnesium acetate tetrahydrate in 100 ml ddH2O and filter with 0.22 micron filter unit

  12. 70% Ethanol

    Note: Solution is freshly prepared before use.

    7.37 ml 190 proof ethanol

    2.63 ml ddH2O

  13. Diffusion buffer (100 ml)

    0.5 M ammonium acetate (5 ml of 10 M ammonium acetate)

    10 mM magnesium acetate (1 ml of 1 M magnesium acetate)

    1 mM EDTA, pH 8.0 (0.2 ml of 0.5 M EDTA, pH 8.0)

    0.1% SDS (0.5 ml of 20% SDS stock)

    ddH2O (93.3 ml ddH2O)

Acknowledgements

Funding sources include the National Institutes of Health (NIH) [F30GM123632 to M.L.H]; Michigan State University DO/PhD Program (to M.L.H.); Intramural Research Program of the NIH, National Institute of Diabetes and Digestive and Kidney Diseases (to M.L.H., A.B.C, L.G.K., and D.M.H.). Protocol is based on the previously published papers (Boulanger et al., 2015; Hsieh et al., 2015; Hsieh et al., 2020).

Competing interests

There are no financial or non-financial competing interests for any of the authors.

References

  1. Boulanger, A., Moon, K., Decker, K. B., Chen, Q., Knipling, L., Stibitz, S. and Hinton, D. M. (2015). Bordetella pertussis fim3 gene regulation by BvgA: phosphorylation controls the formation of inactive vs. active transcription complexes. Proc Natl Acad Sci U S A 112(6): E526-535.
  2. Galas, D. J. and Schmitz, A. (1978). DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res 5(9): 3157-3170.
  3. Hampshire, A. J., Rusling, D. A., Broughton-Head, V. J. and Fox, K. R. (2007). Footprinting: a method for determining the sequence selectivity, affinity and kinetics of DNA-binding ligands. Methods 42(2): 128-140.
  4. Hsieh, M. L., Waters, C. M. and Hinton, D. M. (2020). VpsR Directly Activates Transcription of Multiple Biofilm Genes in Vibrio cholerae. J Bacteriol 202(18): e00234-20.
  5. Hsieh, M. L., Hinton, D. M. and Waters, C. M. (2018). VpsR and cyclic di-GMP together drive transcription initiation to activate biofilm formation in Vibrio cholerae. Nucleic Acids Res. 46(17): 8876-8887.
  6. Maxam, A. M. and Gilbert, W. (1977). A new method for sequencing DNA. Proc Natl Acad Sci U S A 74(2): 560-564.
  7. Sasse-Dwight, S. and Gralla, J. D. (1989). KMnO4 as a probe for lac promoter DNA melting and mechanism in vivo. J Biol Chem 264(14): 8074-8081.

简介

[摘要] DNA足迹是研究蛋白质-DNA相互作用的经典技术。但是,如果蛋白质与DNA的结合较弱,蛋白质的脱落速率较快,或者形成了几种不同的蛋白质-DNA复合物,则传统的足迹方案可能会失败或难以解释。我们的协议不同于传统的足迹协议,因为它提供了一种在使用足迹剂处理后从天然凝胶中分离蛋白质-DNA复合物的方法,从而从游离DNA或其他蛋白质-DNA复合物中去除了结合的DNA。然后从分离的复合物中提取DNA,然后在测序凝胶上电泳以确定印迹模式。该分析为无法使用传统足迹法确定蛋白质与DNA接触的人提供了可能的解决方案。

[背景]核酸酶/化学足迹是一个典型的方法来探测蛋白质-DNA相互作用(腊士和施米茨,1978;萨瑟-德怀特和Gralla,1989;汉普等人,2007) 。在该技术中,蛋白质与DNA特定区域的结合会抑制(保护)或增加(增强)核酸酶或化学试剂切割DNA的能力。因此,如果使用5'端标记的DNA,则通过将蛋白质-DNA复合物与裂解试剂一起孵育,然后在测序凝胶上对DNA进行电泳,可以揭示一种特定的保护/增强模式。但是,为了获得清晰,可解释的模式,蛋白质在切割过程中必须相对紧密地结合DNA。如果利率结合的蛋白(S)的DNA弱和/或如果蛋白质具有快速关闭-速率,传统的足迹方法很可能会失败。此外,有时可以形成具有不同蛋白质-DNA接触的多种复合物。在这种情况下,足迹模式将仅显示交互的完整集合,而不是与离散复合体相关的交互。

在这里,我们已经开发出一种简单的方法来处理具有DNase I或KMnO 4的蛋白质放射性标记的DNA复合物,然后使用电泳迁移率测定(EMSA)分离结合的复合物。在该测定中,将DNA与目标蛋白质孵育,然后通过在天然凝胶上进行电泳,将游离DNA与结合蛋白质的DNA分离。发生这种分离的原因是,蛋白质-DNA复合物迁移的速度比凝胶上的游离DNA迁移慢。然后从切下的聚丙烯酰胺凝胶切片中提取目标蛋白质-DNA复合物,然后分离DNA并在测序凝胶上进行电泳以确定印迹。在许多情况下,即使研究较不稳定和/或弱结合的复合物,该协议也允许用户获得清晰的足迹图案。对于DNase I反应,有或没有蛋白质的保护模式表示蛋白质结合位点,而超敏部位/增强模式表示DNA弯曲,扭结和/或环化。对于KMnO 4反应,增强带代表含有单链胸腺嘧啶残基的区域,例如在开放转录气泡内。然而,该程序可以适用于其他类型的酶或化学试剂,这将产生不同类型的模式。

在实验室中,我们已成功地使用该方案为两个不同细菌系统中的转录激活剂获取了清晰的蛋白质-DNA复合物足迹:百日咳博德特氏菌BvgA (Boulanger等人,2015)和霍乱弧菌VpsR (Hsieh等人,2018)和2020年)中是否存在RNA聚合酶(RNAP)。在VpsR的情况下,我们从游离DNA中分离了在存在或不存在小分子环状di-GMP(c-di-GMP)的情况下形成的蛋白质-DNA复合物(图1A,1 B)复合物本身的直接模式(图1C)。在磷酸化反应调节的情况下BvgA (BvgA〜p ),而只有一个复杂的,开放络合物(RPO ),通过RNA聚合酶在没有形成核糖核苷三磷酸(的rNTPs )(图2A,泳道1对。2),两种复合物,两者RPO和起始复合物(RPI )中,通过在RNAP的存在下形成的rNTPs (图2A,泳道3)。因此,我们使用这种技术来分离RPI从自由DNA和从RPO ,使我们能够分别各可视化复合物的DNA酶I足迹(图2B,泳道2和3)。我们也采用这种方法用KMnO一起4足迹确定内转录气泡的位置RPO通过RNAP与非磷酸化形成BvgA (图2A,泳道4)或内RPO通过RNAP和形成BvgA〜p (图2A,泳道2)。这产生了分别在图2C,泳道1和泳道2中看到的KMnO 4足迹。然后,我们能够将这些结果与没有进行复杂分离步骤得到的结果进行比较(图2D,泳道1和2),从而使我们能够确定稳定的复合物与溶液中形成的复合物的混合物有何不同。





图1. EMSA / DNase I足迹示例。该图已改版并在Hsieh等人的许可下转载。,(B)EMSA凝胶,显示在存在和不存在小分子c-di的情况下,霍乱弧菌调节剂VpsR与霍乱弧菌基因vpsL (P vpsL )的启动子DNA结合形成的复合物如所示,在-GMP以及不存在(A)和存在(B)的RNA聚合酶(RNAP)的情况下。游离DNA的位置由灰色箭头指示,而移位的复合物由黑色箭头指示。结合反应(A)中含有5 nM的32 P标记的非模板P vpsL -97〜103相对于转录起始位点,窝藏1 μ克聚(二DC)作为竞争,和从0至2的量VpsR的增加μ中号为50的情况下(泳道1-5)和存在(泳道6-10)μ中号C-二GMP。结合在(B)反应含有0.04 μ中号P vpsL ,0.14 μ中号重构RNAP(σ :核心的2.5:1的比例),1.4 μ中号VpsR和50 μ中号C-二GMP,所指示的,并且反应与500 ng肝素竞争15秒。(C)测序凝胶,显示来自分离的复合物的DNA酶I产物。在这些情况下,将反应液与0.3 U的DNase I在37 ° C下孵育30 s,然后加载到EMSA凝胶上,将游离的DNA和复合物切下并分离,提取后,将DNA电泳在凝胶上。GA表示G + A阶梯。图像右侧显示-10元素,-35元素和P vpsL +1位置的示意图。黑色矩形框和黑色细箭头分别指示保护模式和超敏部位。虚线红色框表示VpsR结合位点。





图2. EMSA / DNase I足迹和KMnO 4足迹的示例,有和没有从EMSA凝胶中分离出蛋白质-DNA复合物。图已改编并在Boulanger等人的许可下于2015年重新印制。(A)EMSA凝胶显示百日咳博德特氏菌反应调节剂BvgA形成的复合物,磷酸化(BvgA〜P)或非磷酸化(BvgA)的启动子在百日咳博德特氏菌基因FIM3 (P FIM3 ),和RNA聚合酶。结合反应包含0.05 pmol DNA,5 pmol BvgA或BvgA〜P和0.75 pmol重构的RNAP(σ :核心比为2.5:1)。所有样品均用竞争200 μ克/ ml肝素和所指示的,加入的rNTPs(GTP,ATP,和CTP)。游离DNA用灰色箭头表示,而开放复合物(RP O )用黑色箭头标记,而起始复合物(RP I )用虚线黑色箭头表示。(B)测序凝胶,显示与0.36 U的DNase I的结合反应和随后的EMSA分离步骤在P fim3处的DNase I足迹。绑定区域以及-35,-10和+1的示意图位于图像的右侧。用RPi观察到的在-15和-80处的超敏位点,但用RPo观察不到,用'*'表示。(C)和(D)测序凝胶,显示通过在37 ° C下用2.5 mM KMnO 4处理结合反应2.5分钟而获得的P fim3 +1位区域的KMnO 4印迹,然后从EMSA凝胶中获得复合物在不进行EMSA分离步骤(D)的情况下,在运行测序凝胶(C)或直接使用结合反应产物之前分离DNA。结合反应包含0.5 pmol DNA,12 pmol BvgA或BvgA〜P和1.125 pmol重构的RNAP(σ :核心比为2.5:1)。GA代表G + A阶梯。

关键字:DNasel足印分析, KMnO4足印分析, 凝胶阻滞分析, EMSAs, 蛋白质-DNA相互作用

 
材料和试剂
 
注意:除非另有说明,否则所有试剂均应在室温下保存。
1. UltraCruz ®放射自显影带(Santa Cruz Biotechnology公司,目录号:SC-200214)      
2.透明的保鲜膜,例如Saran W rap或Glad Cling Wrap保鲜膜      
3.剃须刀(Personna Gem,目录号:62-0179或同等产品)      
4. 1.7 ml微型管(无菌,无RNase和DNase)(Genesee Scientific,目录号:24-282S)      
5. 0.2 ml PCR管(Axygen ,目录号:PCR-02-C)      
6. 1.5毫升杵(Fisher Scientific,目录号:12-141-364)      
7. 50 ml,0.22微米的Millipore Steriflip过滤系统(Sigma - Aldrich,目录号:SCGP00525)      
8. Ultrafree MC离心过滤器(Millipore,目录号:UFC30HV00 )      
9. 8英寸x 10英寸Hyblot CL自动放射胶片(Denville Scientific,目录号:E3018)      
10. 14英寸x 17英寸Hyblot CL自动放射胶片(Denville Scientific,目录号:E3031)   
11. TranScreen -HE(柯达,目录号:881-1457)   
12. 8英寸x 10英寸胶片盒(国际研究产品,目录号:420180)   
13. 14英寸x 17英寸胶片盒(国际研究产品,目录号:421417)   
14. 8英寸x 10英寸胶片暗盒安全袋(Thomas Scientific,目录号:E3753-1)   
15. 14英寸x 17英寸胶片盒安全袋(Thomas Scientific,目录号:E3753-0)   
16.待测试的纯化蛋白和特定蛋白的适当温度保存   
17.退火至DNA区域5'末端(通常为20-30个碱基对(bp ))的寡核苷酸,用于PCR扩增足迹所需的DNA片段,以50 pmol / μg溶解于ddH 2 O中l并保存在-20 °C (我们建议使用PCR产生200至300 bp的DNA片段。我们从Integrated DNA Technologies获得了寡核苷酸。但是,可以从许多来源购买寡核苷酸。   
18.含有蛋白质结合DNA区域的基因组DNA或质粒DNA,可以用作PCR扩增的模板,以100 ng / ml的浓度溶解于ddH 2 O或TE(参见下文)中,并以4    °C(基因组DNA)或-20 °C (质粒DNA)
19. [ γ - 32 P] -ATP(3 ,000次/毫摩尔,10毫居里/毫升,250    μ次)(珀金埃尔默,目录号:BLU002A;贮存于-20 ℃下)
20. Optikinase (10U / μ升)和10 X Optikinase缓冲液(USB Affymetrix公司,目录号:78334Y;贮存于-20 ℃下)   
21.的Pfu涡轮聚合酶(2.5 U / μ升)和10倍的Pfu缓冲液(安捷伦科技目录号:600252;贮存于-20 ℃下)   
22. dNTP混合物(New England Biolabs ,目录号:N0447,10 mM ;存储在-20 °C )   
23. DNA酶I(2 U / μ升)和10×DNA酶I缓冲液(Life Technologies公司,目录号:AM2222;存储在-2 ℃下)   
24.肝素(Sigma公司- Aldrich公司,目录号:H3149,500 μ克/ ml的溶解在灭菌蒸馏水2 O;储存在-20 ℃下)   
25.聚(脱氧肌苷-脱氧胞苷)(Poly(dI-dC ))酸钠盐(Sigma - Aldrich,目录号:P4929,1 mg / ml溶于ddH 2 O;在-20 °C储存)   
26.尿素超纯(Invitrogen,目录号:15505-050)   
27.凡士林   
28.大型活页夹   
29. 40%的19:1丙烯酰胺:双溶液(Bio-Rad Laboratories,目录号:1610144;储存在4 °C )   
30. 40%37.5:1丙烯酰胺:双溶液(Bio-Rad Laboratories,目录号:1610148;储存在4 °C )   
31.四甲基乙二胺(TEMED)(Bio-Rad实验室,目录号:1610800;在4 °C下储存)   
32. pH 8.0的TE饱和苯酚(Sigma - Aldrich,目录号:77607;在4 °C下储存)   
33.苯酚:氯仿:异戊醇(25:24:1 v / v,pH 7.9 )(Life Technologies,目录号:AM9732;储存在4 °C )   
34. 190标准乙醇(华纳-格雷厄姆公司;在-20 °C下储存)   
35.引出塑料吸管(Fisher Scientific公司,目录号:13-711-27)   
36. GlycoBlue TM共沉淀剂(Invitrogen,目录号:AM9515;在-20 °C储存)   
37. 10x TBE Buffer(Quality Biological,目录号:351-001-131)   
38. 50x TAE Buffer(Quality Biological,目录号:351-008-131)   
39. TE pH 8.0(质量生物学,目录号:351-011-131)   
40. 0.5 M EDTA,pH 8.0 (Quality Biological,目录号:351-027-101)   
41.甲酰胺(西格玛- Aldrich公司,猫考勤号码:47670)   
42. 1-丁醇(Fisher Scientific,目录号:A399-500)   
43. 3 M醋酸钠(Sigma - Aldrich,目录号:71196)   
44.干冰   
45.用于天然凝胶的6x染料加载溶液(Fermentas ,目录号:R0611)   
46.二甲苯氰(XC)染料(Sigma - Aldrich,目录号:X4126)   
47.溴酚蓝(BPB)染料(Sigma - Aldrich,目录号:B0126)   
48. AG ® 501-X8(d)混合床树脂(Bio-Rad实验室,目录Ñ棕土:1426425)   
49.鲑鱼精子DNA(Sigma - Aldrich,目录号:D1626; 10 mg / ml,5 mg / ml和1 mg / ml溶于ddH 2 O;在-20 °C下保存)   
50. ddH 2 O   
51. APS(Bio-Rad实验室,目录号:1610700)    
52.醋酸铵(Sigma - Aldrich,目录号:A7330)    
53.氯化钙二水合物(西格玛- Aldrich公司,目录号:C3306)    
54.高锰酸钾4 (西格玛- Aldrich公司,目录号:223468 )    
55.四水合醋酸镁(Sigma - Aldrich,目录号:M5661)    
56.甲酸库存(88%)(Sigma-Aldrich,目录号:399388)。在ddH 2 O中稀释4%(请参阅食谱)    
57. 50 mM KMnO 4 (高锰酸钾)(请参阅食谱)    
58. 10%过硫酸铵(APS)(请参阅食谱)    
59. 10 M醋酸铵(请参阅食谱)    
60. 1 M醋酸镁(请参阅食谱)    
61. 20%SDS(质量生物学,目录号:351-066-721);1%SDS稀释在dd H 2 O中(请参阅食谱)    
62. 2 mM CaCl 2 (氯化钙)(请参阅食谱)    
63.哌啶储备液(10 M)(Sigma-Aldrich,目录号:104094);在ddH 2 O中稀释2 M和100 mM (请参阅食谱)    
64.甲酰胺负载染料;储存在-20 °C (请参阅食谱)    
65. 2-巯基乙醇储备液(14.3 M)(Sigma - Aldrich,目录号:M3148)。在ddH 2 O中稀释1 M (请参见配方)    
66. 70%乙醇(请参阅食谱;在-20 °C下储存)    
67.扩散缓冲液(请参阅食谱)    
 
设备
 
适用于32 P放射性的工作空间
盖革计数器监测放射性和污染
有机玻璃防护罩可保护用户免受放射性伤害
用于32 P废物的有机玻璃盒子
150 ml,0.22微米过滤器设备(Nalgene,目录号:125-0020或同等产品)
PCR机
Vortex(例如Vortex Genie 2,Scientific Industries)
加热块可以加热到95 ° C
垂直凝胶盒-中等大小(〜7” W X〜9” H) (如科尔-帕克垂直单可调平板凝胶系统,目录号:EW-28570-00或Hoefer说TM风冷垂直电泳单位,目录号:SE400 )
垂直凝胶箱-大尺寸(〜14” W X〜17” H) (如LABRepCo模型S2测序凝胶电泳装置中,目录号:21105036或BTLab系统核酸测序电泳细胞(330 X 420毫米),目录号:BT210 )
Elutrap电洗脱系统(GE Healthcare,目录号:10447711)
电泳电源盒(Bio-Rad实验室)
小型微量离心机,例如Benchmark MyFuge Mini,可在6,000 rpm / 2,000 xg的转速下旋转
台式微量离心机,如Eppendorf离心机5425,可以在旋转≥ 14000 ×g下(微量离心,目录Ñ棕土:5405000646)
速度真空(瓦特E使用热电Electron公司萨文特DNA 120 SpeedVac中的系统,其速度是1600转;热电飞世尔科技,产品目录号:13442549)
闪烁计数器
胶片显影剂(或phosphoimager )
密度计扫描放射自显影[瓦特ë使用GS-800校准的成像密度计从Bio-Rad实验室,目录号:170-7983(停产); 新型号是GS-900,目录号:170-7989 ]
带有垫片和梳子的玻璃板,适合中型凝胶设备(Gel Company;我们建议凝胶厚度为1 mm)
带有垫片和梳子的玻璃板,适合大型凝胶设备(Gel Company;我们建议凝胶厚度为1 mm)
 
软件
 
软件来操作密度(我们使用数量一从Bio-Rad实验室,目录号:1709601)
 
程序
 
除非另有说明,否则所有反应均在冰上组装,并在室温下进行离心。
当使用32 P时,请使用安全规程处理放射性,检查表面是否受到污染以及废物处置。我们建议使用不含RNase / DNase的无菌ddH 2 O溶液,并采取适当的预防措施以最大程度地减少DNase污染的可能性。
 
使5 ' - 32 P DNA末端标记任一上的非模板或模板链
激酶反应:5'- 32 P末端标记模板或非模板链寡核苷酸。
在0.2 ml PCR试管中,合并以下各项:
7 μ升[ γ - 32 P] -ATP
1 μ升Optikinase酶
1 μ升10X Optikinase缓冲
1 μ升的寡核苷酸(50皮摩尔/ μ升)
在37 ° C下孵育30分钟。通过在65 ° C下孵育10分钟来灭活酶。
组装PCR反应。
在激酶反应中添加以下内容:
76 μ升的DDH 2 ö
10 μ升10X的Pfu缓冲
1个μ升10毫dNTP混合物
1 μ升质粒DNA或gDNA的(100毫微克/毫升)
1 μ升其它(非标记)寡核苷酸(50皮摩尔/ μ升)
1 μ升的Pfu涡轮酶(2.5 U / μ升)。
通过以下循环进行PCR生成标记的DNA片段:
95 ° C 2分钟使DNA变性
95 ° C 30秒
(m-5 ° C)*的温度持续30 s
72 ° C 30秒
重复步骤bd 25-35个循环
72 ° C 10分钟
4 ℃储存。
* m-5 ° C –比最低引物熔化温度低5 ° C的温度。
前一天,为中型凝胶仪准备4%聚丙烯酰胺凝胶,以确保凝胶完全固化并提高分离度。
使用凡士林组装玻璃板,以密封侧面和底部垫片和大号活页夹的角,从而将玻璃板固定在一起。
掺在一起:
4 ml 40%丙烯酰胺:双19:1
34.8毫升ddH 2 O
0.8毫升50 x TAE
0.4毫升10%APS
通过150毫升0.22微米的过滤器过滤溶液。添加12 μ升TEMED到滤液中,轻轻涡旋混合(避免引入气泡),并立即倒入凝胶和插入梳子。用保鲜膜小心地盖住梳子区域。在凝胶顶部的保鲜膜上使用粘合剂夹,以在玻璃板和梳子之间形成紧密密封。让凝胶在室温下固化过夜。
在4%垂直聚丙烯酰胺凝胶上电泳PCR生成的标记DNA片段。
将凝胶放入中等大小的凝胶仪器中。移开梳子后,用永久性标记物标记玻璃板前面的孔位置。(这显着改善了加载过程中的插槽可视化。)将凝胶在100 V的1x TAE中预运行1 h。将6倍染料加载溶液添加到PCR样品体积中。将所有样品上样到凝胶上。在1x TAE中以140 V运行,直到6x加载染料的BPB组分距起点约13 cm(约2 h)。
从凝胶中消费已标记的DNA。
拆除凝胶。卸下顶部玻璃板。用保鲜膜包裹底板/凝胶。用放射自显影带标记塑料包裹的凝胶的角。请遵循有关标记磁带的说明。将凝胶暴露在薄膜上几分钟。(大约2-3分钟就足够了。)冲洗胶片并通过将胶片与仍包裹的凝胶对齐来确定放射性标记DNA在凝胶上的位置。使用剃须刀在显影的胶片上切出所需的标记的DNA带,以制成模具。将模板对准塑料包裹的凝胶。使用模板,用永久性标记在塑料包装的凝胶上标记标记的DNA的位置。带有剃须刀的消费品乐队。对每个样品使用干净的剃刀。从凝胶上切下标记的DNA后,一定要除去保鲜膜。移开切片时注意凝胶的方向(-与+末端)。
通过电洗脱分离标记的DNA 。
按照说明手册组装Elutrap 。用½X TAE填充室。以与凝胶电泳相同的方向加载切下的标记DNA。(确保凝胶的“ –”端朝向Elutrap的“ –”端,凝胶的“ +”端朝向Elutrap的“ +”端。)在200 V下电泳1 h。收集含有标记DNA的溶液,并将其放入1.7 ml微管中。重复电泳,收集溶液,然后将其添加到同一管中。
沉淀标记的DNA。
Evapo速率标记的DNA溶液至约200 μ升的速度真空。(注意:这可能需要一个小时或更长时间;请使用环境温度设置,并且不要打开热量以加快干燥速度)。加入200 μ升酚(TE饱和的)。混合30 s,在小型微量离心机中以2,000 xg离心1分钟,然后将顶层(水层)中的标记DNA转移至干净的微管中。将5x体积的190标准乙醇和1/4体积的10 M乙酸铵添加到含有标记DNA的苯酚萃取水层中,充分混合,然后在干冰上孵育1 h或在-20 ° C过夜。离心机在台式微量离心机在最高速度(≥ 14000 ×g下)30分钟,用拉出吸管除去乙醇溶液中,并洗涤沉淀的标记的DNA沉淀与100 μ升冰冷的70%乙醇。如上离心5分钟,除去70%乙醇,并在室温下以高速真空干燥标记的DNA 2-3分钟。重悬沉淀在20 μ升TE。这假定32 P掺入DNA的10%至40%,产率> 25%。然而,如果需要,掺入速率和产率可以通过使用TCA(定量三氯酸)沉淀(小号ee值这里)。
 
准备一个G + A测序阶梯(Maxam and Gilbert,1977)
在1.7 ml微量管中,合并以下内容:
1点μ升标记的DNA
1 μ升1mg / ml的鲑鱼精子DNA
8 μ升TE
加入1和μ升4%甲酸(见ř ecipes)。
在37 °C下孵育45分钟。
置于冰上管,加入150 μ升的2M哌啶(见ř ecipes)。
在90 °C下孵育30分钟。
置于冰上管,并加入5 μ升10毫克/毫升鲑鱼精子DNA。
加入1毫升1-丁醇,涡流以充分混合,然后离心在台式微量离心机在≥ 14000 ×g离心5分钟。小心取出并丢弃上清液。
注意:由丁醇沉淀形成的丸粒有从试管侧面浮起的趋势,因此确保除去上清液时不要丢弃丸粒,这一点很重要。
添加150 μ升1%SDS于粒料(见ř ecipes)。
加入1ml丁醇,涡流以充分混合,然后离心在台式微量离心机在≥ 14000 ×g离心5分钟。小心取出并丢弃上清液。
洗涤沉淀两次用0.5毫升丁醇,离心1分钟在台式微量离心机在≥ 14000 ×g下每次洗涤后。小心取出并丢弃上清液。
在室温下以vac-vac干燥沉淀1-2分钟。
添加10 μ升甲酰胺负载溶液溶解并储存在-20 ℃下。
 
进行DNase I或KMnO 4反应
注意:蛋白质和特异性结合缓冲液的量将取决于所用蛋白质。首先,请使用您知道蛋白质活跃的条件或参阅Boulanger等人中列出的条件。(2015)和Hsieh等。(2018和2020)。
DNase I反应
在总共10 μ升体积,感兴趣孵育蛋白(或作为阴性对照只是蛋白质的缓冲液),标记的DNA,并且所述蛋白质结合到DNA结合缓冲液为宜。此外,缓冲液应含有2 mM CaCl 2以用于DNase I活性。温度应适合所测试的蛋白质,但通常在室温至37 °C之间。我们建议每个反应使用0.1至0.5 pmol的DNA。之比亲TEIN:DNA可根据被测试什么条件来改变。为了获得良好的结合,我们建议至少要比标记的DNA多5至10倍过量的蛋白质。为了消除不稳定和非特异性复合物,我们也建议在加入1的μ升1毫克/毫升的聚(DI-DC )在结合反应或0.5至1 μ升的500 μ克/ ml肝素的结合反应之后完成了。稀释股票DNA酶I酶(2U / μ升在1×DNA酶I)缓冲剂配制至所需浓度。我们建议尝试一定范围的DNase I浓度。我们以前总共使用了约0.3U。以引发DNA酶I反应,加1 μ升稀释DNA酶I酶(或1 μ升1×DNA酶I缓冲液作为对照)。最终反应体积为11 μ升。反应也可以扩大规模。通过用手指在试管上轻敲2-3次来混合反应成分,然后使用小型微量离心机以2,000 xg的速度快速离心样品。该步骤仅需约15 s。将样品置于37 ° C加热块中,孵育30 s。(如果需要更多或更少的裂解,可以改变这个时间。)立即将样品加载到已经以100 V / h运行的凝胶上。参见下面:步骤D3 。
高锰酸钾4 reactio NS
感兴趣孵育蛋白(或作为阴性对照只是蛋白质的缓冲液中)与一个标记的DNA适当的结合缓冲液(10 μ升在37总体积)° C至开始形成的DNA的单链区,诸如在使用RNAP时的开放转录复合体。我们建议使用0.1至0.5 pmol的标记DNA。在该温育,竞争对手,诸如肝素(1之后μ升的500 μ克/ ml溶液),可与约1分钟的额外温育被添加到除去不稳定的复合物。添加0.5 μ升50毫的KMnO 4 (参见ř ecipes)(或0.5 μ升的的DDH 2 O作为阴性对照)。确保在使用当天制作KMnO 4溶液。温育在37 2.5分钟° C.淬灭反应,加入5 μ升的1M 2-巯基乙醇(见ř ecipes)。立即将样品加载到已经以100 V / h运行的凝胶上。参见下文:步骤D3。
 
在4%丙烯酰胺天然凝胶上电泳DNase I处理或KMnO 4处理的复合物
前一天准备用于大型凝胶设备的4%聚丙烯酰胺凝胶,以确保凝胶完全固化并提高分辨率。
使用凡士林组装玻璃板,以密封垫片和大的活页夹的角,以将玻璃板固定在一起。
掺在一起:
12 ml 40%丙烯酰胺:bis 37.5:1
12毫升10x TBE
95.28毫升ddH 2 O
0.72毫升10%APS。
通过150毫升0.22微米过滤器过滤溶液。除去4毫升凝胶溶液中,添加4 μ升TEMED该等分试样,拌匀,并倒入平板。一旦为插头的凝胶固化的该部分(〜15分钟),加入20 μ升TEMED剩余凝胶溶液,通过反转拌匀,但不要引入气泡。立即倒入盘子。轻敲玻璃板上的气泡以清除所有气泡。将其水平放置在支架上,然后立即插入梳子。用保鲜膜小心地盖住梳子区域。在凝胶顶部的保鲜膜上使用粘合剂夹,以在玻璃板和梳子之间形成紧密密封。使凝胶在室温下固化过量。
在100 V / h下预电泳2小时。(请确保使用永久性标记在玻璃板正面的孔位置上进行标记。这显着改善了装载期间的插槽可视化。)
当凝胶以100 V / h的速度运行时,在一个泳道中加载一个反应的所有样品。为了确保良好的分离,在加载每个新样品时跳过一个泳道。(如果需要更多样品,可以将相同的反应进行多次并加载到多个泳道中。在切下凝胶切片后将产物合并。)
在380 V下运行凝胶3小时。
拆除凝胶。取下顶板,并用保鲜膜包裹凝胶/底部板。
 
从4%丙烯酰胺天然凝胶中分离并提取ct标记的DNA
用放射自显影带标记塑料包裹的凝胶的每个角。这将有助于模板对齐。
将未曝光的胶片放在暗室胶卷暗盒中的凝胶上。
将胶片曝光过夜。
冲洗胶卷。
详见在步骤A6使模版,但是在这种情况下,切出的游离的标记的DNA条带,并从膜中任何所希望的配合物两者。采取获得所有放射性所需的最小量的凝胶。切片的宽度通常约为4-5毫米(凝胶泳道的宽度),长度约为3毫米。但是,如果需要,可以采用更长的切片。乙Ë确保标签。
将每个凝胶切片放入一个空的1.7 ml微型管中。
在微管中用1.5毫升的杵粉碎聚丙烯酰胺凝胶。
加入200 μ升扩散缓冲液(见ř每切片带ecipes)。(如果将多个切片合并,则添加适当量的扩散缓冲区。)
将试管在60 ° C下孵育至少2 h或在室温下孵育过夜。
离心机在台式微量离心机在≥ 14000 ×g离心5分钟。
用移液器吸头倾析或小心除去溶液。
将溶液转移至MC离心过滤器。
离心在台式的MC离心过滤器的离心在≥ 14000 ×g离心5分钟以除去聚丙烯酰胺的痕迹。
将流通液转移到干净的1.7 ml微管中。
在高速真空,减少体积至约200 μ升对于DNA酶I反应和〜100微升为的KMnO 4个反应,如果必要的话。
注意:这可能需要一个小时或更长的时间;请使用环境温度设置,请勿打开热量以加快干燥。
对于DNase I反应,可通过苯酚提取溶解。加入200 μ升苯酚:氯仿:异戊醇,在小混合30秒,离心分离机的离心在2000 ×g下进行1分钟,和含有该标记的DNA的顶部(水溶液层)转移到一个干净1.7毫升微型管。
乙醇沉淀DNA。加入5倍体积的190标准乙醇,10μM的乙酸铵¼DNA体积(见ř ecipes),和1 μ升GlycoBlue在干冰上苯酚萃取水层DNA,拌匀,孵育1个小时或在-20 °下过夜,离心分离机在台式微量离心机在≥ 14000 ×g离心30分钟,除去乙醇/醋酸铵/ GlycoBlue与引出吸管溶液,洗涤标记的DNA沉淀与100 μ升冰冷的70%乙醇,离心如上5分钟,除去70%的乙醇,并在室温下真空高速干燥2-3分钟。
在F或KMnO 4反应中,乙醇沉淀标记的DNA。在-20添加10倍体积的190标准乙醇,搅拌均匀,孵化°下进行20分钟,在离心机台式微量离心机在≥ 14000 ×g离心20分钟,用拉出吸管除去乙醇溶液,洗标记的DNA沉淀与100 μ升冰冷的70%乙醇,离心分离机在台式微量离心机在≥ 14000 ×g离心5分钟,除去70%乙醇,并在高速真空干燥2-3分钟。重悬沉淀在100 μ升100毫哌啶(见ř ecipes)孵育,在90 °下进行30分钟,以进行裂解反应。之前置于冰上5分钟,将样品在加入2的μ升5毫克/毫升鲑鱼精子DNA的。加入1毫升1-丁醇,涡旋样品以充分混合,并离心在台式微量离心机在≥ 14000 ×g离心5分钟(注意:由丁醇沉淀形成的颗粒具有从管的一侧漂走的倾向,因此是重要确保除去上清液当团粒是不被丢弃。) 。W¯¯灰与10 μ升1-丁醇,自旋在台式微量离心机在≥ 14000 ×g离心5分钟,并移除丁醇洗涤。在高速真空下干燥样品2-3分钟。重悬沉淀在18.75 μ升TE和6.25 μ升3M乙酸钠。如步骤17所述,重复进行乙醇沉淀。
重悬在10粒料μ升甲酰胺上样染料(见ř ecipes)。
在闪烁计数器中计数样品。
 
在8%变性测序凝胶和图像凝胶上电泳标记的DNA和G + A阶梯
前一天准备针对大型凝胶设备的8%变性变性测序凝胶,以确保凝胶完全固化并提高分离度。
使用凡士林组装玻璃板,以密封垫片和大的活页夹的角,以将玻璃板固定在一起。
合并在250毫升的烧杯中:
24 ml 40%丙烯酰胺:双19:1
53.3克尿素
50.3毫升ddH 2 O
1克混合床树脂
将烧杯置于37 ° C直至所有尿素溶解(这可能需要≥20分钟)。混合床树脂将保留。旋转混合,然后通过150毫升0.22微米过滤器过滤。向滤液中加入6 ml 10x TBE和0.72 ml 10%APS。通过加入4倒入凝胶“插头” μ升TEMED至4毫升上述凝胶溶液。充分混合并倒入玻璃板中。等待固化(〜15分钟)。添加20 μ升TEMED剩余凝胶溶液,以及通过反转混合,但不引入气泡。立即倒胶。轻敲玻璃板上的气泡。将水平放置在支架上,然后立即插入梳子。用保鲜膜小心地盖住梳子区域。在凝胶顶部的保鲜膜上使用粘合剂夹,以在玻璃板和梳子之间形成紧密密封。让凝胶在室温下固化过夜。
在500V进行预运行凝胶≥ 30分钟,750伏为≥ 30分钟,然后1 ,000 V为≥ 30分钟以½ X TBE。
注意:请确保使用永久性标记在玻璃板前面的孔位置进行标记。这显着改善了加载期间的插槽可视化。
停止运行前凝胶。在95 ° C下加热样品2分钟。在这段时间内,使用抽出的移液器用运行缓冲液冲洗每个孔,以除去扩散到孔中的所有尿素。
加载样品(以及G + A梯道以确定DNA的序列)。在每个通道中加载大约相同的cpm (基于闪烁计数器值)。我们建议每个泳道至少加载8,000个计数。可以使用较少的量,但需要延长胶片的曝光时间。
在1运行凝胶,000 V /为〜3小时小时。(确切的时间将取决于标记的DNA区域,该区域需要具有最大的分辨率才能观察到足迹)。
从设备上去除凝胶。取下顶板,并用保鲜膜包住凝胶/底部板。
将未曝光的胶片放在暗室中的塑料包装的凝胶/底板上,并将凝胶板/凝胶/胶片存放在放在-80 ° C冰箱的暗盒安全袋中的暗盒中。为了帮助减少静电,您还可以在未曝光的胶片和塑料包裹的凝胶之间添加一个额外的曝光的胶片。可替代地,可以代替地使用磷光体成像屏幕和磷光体成像器。
所需的天数后,从凝胶上除去薄膜并显影。如果需要其他曝光,请添加新胶卷。如果信号微弱,并且需要信号放大,请考虑使用TranScreen 。
在密度计上扫描放射自显影照片。我们使用Bio-Rad Laboratories的GS-800密度计。
 
菜谱
 
我们建议使用不含RNase / DNase的无菌ddH 2 O溶液,以最大程度地减少RNase或DNase污染的可能性。除非另有说明,否则溶液是在室温下制备和储存的。
4%甲酸
9.1 μ升88%库存加上190.9的μ升的DDH 2 ö
注:中号使用前阿克新鲜的权利。
1%SDS
7.5 μ升的20 %SDS库存加上142.5 μ升的DDH 2 ö
10%APS
1克过硫酸铵溶于10毫升ddH 2 O
溶液在使用前新鲜配制。
甲酰胺加载染料
混合1毫升去离子甲酰胺和10 μ升XC的饱和溶液和10 μ升饱和溶液BPB。溶液可以在-20 °C下保存1周。
注意:要的去离子甲酰胺,加入约0.5克AG ® 501-X8(d)的混合床树脂到5ml的甲酰胺使用0.22微米微孔在室温下,温育至少10分钟,和过滤器Steriflip 。要制成XC或BPB的饱和溶液,请向无菌的1.7 ml试管中加入约0.2 g的染料,并加入1 ml的ddH 2 O,剧烈涡旋,并使用小型微量离心机以2,000 xg的速度进行短暂离心,以沉淀未溶解的粉末。除去10 μ升从剩余的粉末上方的液体添加到升ö ading色素溶液。
10 M醋酸铵
将77 g乙酸铵溶于100 ml ddH 2 O,并用0.22微米过滤器过滤
2毫米CaCl 2
将0.029 g脱水氯化钙溶于100 ml ddH 2 O中,并用0.22微米过滤器过滤
50毫米KMnO 4
对于250 mM的储备液,将3.95 g KMnO 4溶解在100 ml ddH 2 O中
注意:可以在室温下保存数周。
对于50毫米的KMnO 4 ,应在使用的当天新鲜制,混合10 μ升250毫米的KMnO 4与40 μ升的DDH 2 ö
100 mM哌啶
注意:溶液在使用前是新鲜配制的。
10 μ升的哌啶股票(10 M)
990 μ升的DDH 2 ö
2 M哌啶
注意:溶液在使用前是新鲜配制的。
30 μ升的哌啶原料(10 M)
120 μ升ħ 2 ö
1 M 2-巯基乙醇
注意:溶液储存在-20 °C ,可解冻/冷冻数月。
7 μ升的2-巯基乙醇的库存(14.3 M)
93 μ升的DDH 2 ö
1 M醋酸镁
将21.45 g四水合乙酸镁溶解在100 ml ddH 2 O中,并用0.22微米过滤器过滤
70%乙醇
注意:溶液在使用前是新鲜配制的。
7.37毫升190标准乙醇
2.63毫升ddH 2 O
扩散缓冲液(100毫升)
0.5 M醋酸铵(5 ml 10 M醋酸铵)
10 mM醋酸镁(1 ml 1 M醋酸镁)
1 mM EDTA,pH 8.0(0.2 ml的0.5 M EDTA,pH 8.0)
0.1%SDS(0.5 ml的20%SDS库存)
ddH 2 O(93.3 ml ddH 2 O)
             
Acknowled克È换货小号
 
资金来源包括美国国立卫生研究院(NIH)[F30GM123632至MLH];密歇根州立大学DO / PhD计划(MLH);美国国立卫生研究院(NIH)的壁内研究计划,美国糖尿病与消化,肾脏疾病研究所(针对MLH,ABC,LGK和DMH)。协议基于先前发表的论文(Boulanger等,2015; Hsieh等,2015; Hsieh等,2020)。
 
利益争夺
 
对于任何作者来说,都没有经济或非金融竞争利益。
 
参考文献
 
Boulanger,A.,Moon,K.,Decker,KB,Chen,Q.,Knipling ,L.,Stibitz ,S. and Hinton,DM(2015)。百日咳博德特氏菌FIM3基因调控由BvgA :磷酸化控制无活性与活性转录复合物的形成。PROC国家科科学院科学USA 112(6):E526-535。
Galas,DJ和Schmitz,A。(1978)。DNAse足迹:检测蛋白质-DNA结合特异性的简单方法。 Nucleic Acids Res 5(9):3157-3170。
汉普郡(AJ),鲁斯林(Rusling),DA,布劳顿-海德(Broughton-Head),VJ和福克斯(KR)(2007)足迹:一种确定DNA结合配体的序列选择性,亲和力和动力学的方法。方法42(2):128-140。
Hsieh,ML,Waters,CM和Hinton,DM(2020)。VpsR直接激活霍乱弧菌多个生物膜基因的转录。Ĵ细菌学202(18):e00234-20。
Hsieh,M. L.,Hinton,DM和Waters,CM(2018)。VpsR和环状双GMP共同驱动转录起始,以激活霍乱弧菌的生物膜形成。核酸研究。46 (17):8876-8887。
Maxam,AM和Gilbert,W。(1977)。DNA测序的新方法。美国国家科学院院刊74(2):560-564。
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引用:Hsieh, M., Boulanger, A., Knipling, L. G. and Hinton, D. M. (2020). Combining Gel Retardation and Footprinting to Determine Protein-DNA Interactions of Specific and/or Less Stable Complexes. Bio-protocol 10(23): e3843. DOI: 10.21769/BioProtoc.3843.
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