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This ChIP protocol was developed and improved over the years by various researchers in the Snyder lab, Stanford University, especially Anthony Borneman and Christopher Yellman. I have used this method to successfully map the genome-wide binding of transcription factors Ste12. The ChIPed DNA is suitable for downstream analysis using PCR, microarray or sequencing.

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[Bio101] Yeast Transcription Factor Chromatin Immunoprecipitation
[Bio101] 酵母转录因子的染色质免疫沉淀

分子生物学 > DNA > DNA-蛋白质相互作用
作者: Wei Zheng
Wei ZhengAffiliation: Keck Biotech Services, Yale University, New Haven, USA
For correspondence: wei.zheng.madison@gmail.com
Bio-protocol author page: a10
7/5/2011, 7127 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.92

[Abstract] This ChIP protocol was developed and improved over the years by various researchers in the Snyder lab, Stanford University, especially Anthony Borneman and Christopher Yellman. I have used this method to successfully map the genome-wide binding of transcription factors Ste12. The ChIPed DNA is suitable for downstream analysis using PCR, microarray or sequencing.

[Abstract] 本篇 ChIP 的方法是Snyder实验室多年来经多位学者改进优化而来,特别是 Anthony Borneman 和 Christopher Yellman。我应用此方法已成功定位了转录因子Ste12在基因组范围内的结合。ChIP得到的DNA可通过PCR、芯片及测序进行下游的分析。

Materials and Reagents

  1. 500 ml of log-phase yeast cell culture per ChIP (at -0.9 x 107 cells/ml, ~4.5 x 109 cells per sample)
  2. 37% formaldehyde
  3. 2.5 M glycine in H2O (heat sterilized)
  4. Liquid nitrogen, dry ice/ethanol bath or -70 °C freezer
  5. 0.5 mm Zirconia/Silica Beads (Bio Spec Products, catalog number: 11079105z )
  6. Commercial protease inhibitor cocktails, for example:
    Roche Complete protease inhibitor cocktail tablets (F. Hoffmann-La Roche, catalog number: 11697498001 )
    Roche Complete Mini protease inhibitor cocktail tablets (F. Hoffmann-La Roche, catalog number: 11836153001 )
  7. EZview anti-Myc affinity gel (red colored beads) (Sigma-Aldrich, catalog number: E6654 )
  8. Minelute kit for final DNA purification (QIAGEN, catalog number: 28004 )
  9. LiCl
  10. NaOAc
  11. Ethanol
  12. Triton X-100
  13. TE
  14. NaCl
  15. EDTA
  16. Isopropanol
  17. NP-40
  18. Na-deoxycholate
  19. SDS
  20. Tris-buffered saline (TBS) (10x stock) (see Recipes)
  21. Lysis/IP buffer (see Recipes)
  22. Lysis buffer/500 mM NaCl (see Recipes)
  23. IP wash solution (see Recipes)
  24. TE/1% SDS (100 ml) (see Recipes)
  25. TE/0.67% SDS (100 ml) (see Recipes)
  26. TE (100 ml) (PH 8.0) (see Recipes)
  27. 1 mM PMSF (Fluka, catalog number: 93482 ) (see Recipes)

Equipment

  1. Millipore stericup sterile vacuum filter units, 500 ml funnel, 0.22 μm or 0.45 μm pore size (EMD Millipore, catalog number: SCGVU05RE , SCHVU11RE )
  2. Syringe needle (BD Biosciences, catalog number: 305155 or 305156 )
  3. 5 ml snap-cap tubes, polypropylene (preferred) or polystyrene (BD Biosciences, Falcon®
  4. 15 and 50 ml conical polypropylene screw-top tubes (BD Biosciences, Falcon®)
  5. Branson Sonifier 250 with microtip or Digital Sonifier S-450D (BD Biosciences)
  6. Refrigerated tabletop centrifuges, e.g. Beckman GS-6R, GS-15R (Beckman Coulter), or Eppendorf refrigerated multipurpose centrifuges (EppendorfTM, model: 5810R and 5804R ) (or simply put an ordinary tabletop centrifuge in the cold room) 
  7. Fume hood
  8. FastPrep machine (FastPrep, catalog number: 6004500 )
  9. Hemacytometer
  10. Spectrophotometer

Procedure

Day 0

  1. Set up the experiment.
    Each IP is from 500 ml of cells in mid-log phase at OD600 of ~0.6, a density of ~0.9 x 107 cells/ml. The total number of cells per IP is ~4.5 x 109, and the total cell weight per sample should be 0.2-0.25 g.
    Note: Other ChIP protocols specify 100 ml of cells at 107 cells/ml, or 109 total cells. If in doubt about cell number (for example when dealing with clumpy yeast strains), use a hemacytometer to count cells instead of using the spectrophotometer.
  2. Grow cells under the desired conditions
    Growth conditions for inducing pheromone response transcription are described in Zheng et al. (2010).
  3. Treat the cells with formaldehyde to crosslink proteins and DNA
    In a fume hood, add 37% formaldehyde to the cells to a final concentration of 1% (use 14 ml formaldehyde). Maintain the cells at room temperature (RT) for 15 min, swirling occasionally to mix. Effective fixation conditions vary according to the protein that will be immunoprecipitated. The simplest way to optimize this variable is to change (in most cases increase) the fixation time.
  4. Quench the crosslinking reaction with glycine
    Add 2.5 M glycine to a final concentration of 125 mM (a 20x dilution, so add 27 ml). Incubate the samples for 5 min at RT with occasional mixing.
  5. Collect and wash the cells
    Collect the cells by filtration using a 0.45 or 0.22 μm filter. Wash the cells twice on the filter with 100 ml of water at RT. Rinse the cells from the filter using 20 ml of water and transfer them to a 50 ml polypropylene tube. Repeat the rinse to collect residual cells.
    Spin at 4,000 rpm for 5-25 min to pellet the cells and discard the supernatant. Resuspend the cells in 1 ml of water and transfer them to a 2 ml screw cap tube (for later lysis). Spin the cells down in a microcentrifuge (3 min at max rpm) and thoroughly remove the supernatant. Weigh the samples at this point. Each 500 ml culture should yield 0.2-0.25 g of cells. Add 1 ml of zirconium beads to each sample to prepare it for the lysis step. Keep the samples on ice or freeze them for storage. Process the experimental replicates separately from here forward.
    Notes:
    1. It is a good idea to check the total weight of cells recovered. Cell weight can be strain dependent and differs significantly between haploids and diploids.
    2. The cells can be kept on ice for several hours at this point or frozen for storage. Putting the samples directly into a -70 °C freezer works well.

Day 1

  1. Prepare lysis/IP buffer with protease inhibitors
    Prepare 6 ml of lysis/IP buffer for each sample of cells and some extra for equilibrating the antibody beads (50 ml for 6 ChIPs works). Use Roche complete protease inhibitor pellets, which will treat 50 ml of buffer. When using the tablets, it is still necessary to add PMSF. Add 0.75 ml of lysis/IP buffer with protease inhibitors to each sample tube.
    Note: It is best to add PMSF last, just before using the buffer, since it is unstable in aqueous solutions, with a half-life of ~35 min at pH 8.
  2. Lyse the cells with cubic zirconium beads
    Perform all manipulations in an ice/water bath. Disrupt the cells with the FastPrep machine, using a total of five 1 min bursts at speed 6.0 (additional rounds only if needed). After each burst, immerse the samples in ice water for a minute or so to keep them cold.
    Note: Examine the cells under the microscope to check for effective lysis. The number of lysed cells should approach 100%.
  3. Recover the crude lysate
    Prepare a Falcon Sml snap-cap tube for each sample. Place the 2 ml lysis tube top-down on the benchtop. Heat a syringe needle to red hot in a flame and use it to pierce the bottom of each sample tube. Put the 2 ml tube about 1 cm into a 5 ml Falcon tube, where it should rest snugly. Centrifuge in a tabletop (preferably chilled) for 1 min at 1,500 rpm, bringing the lysate down into the Falcon tube. Add 0.75 ml of cold lysis buffer to each tube to wash the beads and spin again. Transfer the lysate to a 15 ml conical tube (good for sonication), and add 2.4 ml of lysis buffer. Total lysate volume should be ~4 ml.
  4. Sonicate the lysate to shear chromatin
    Shear the chromatin by sonicating the suspension with a Branson 2S0 Sonifier fitted with a microtip. Use the sonifier at amplitude 6 and 100% duty cycle. Sonicate each sample 5 times for 30 sec each time. Hold the tube in a small beaker of ice/water while sonicating. Between sonications chill the samples in ice/water for at least 2 min. Also chill the sonifier tip in ice water periodically (after 18x sonications) to keep it from getting too hot. If using the Digital Sonifier S-450, use 15 times for 10 sec each to avoid overheating. Set total run time as 2 min 30 sec, amplitude 50%, pulse on 10 sec, pulse off 1 min. Hold the tube in a small beaker of ice/water. Preferably the whole procedure is done in a cold room. The average length of DNA post-sonication should be 500 bp, with a range of 100-1,000 bp.
    Notes:
    1. Sonication should be monitored and adjusted to yield the desired average DNA length as described in Notes section.
    2. Clean the sonicator tip after use.
    3. A suggested routine is to dip the probe in 0.1 % SDS, then water, spray it with ethanol and dry it with a kimwipe tissue.
  5. Remove cell debris from the lysate by centrifugation
    Centrifuge the lysates at 3,000 rpm in a refrigerated tabletop centrifuge for 5 min at 4 °C, remove the supernatant and divide it into two 2 ml microcentrifuge tubes. Spin in a cooled microcentrifuge at 14,000 rpm for 10 min, remove the supernatants, and pool the two lysates into a fresh 15 ml conical tube. The lysates are now ready to use for IP, and one can save aliquots at this point for analysis of total chromatin and protein. This is often used as input control.
    Note: Avoid carrying over any aggregated debris by staying away from the pellet, sacrificing ~50 μl of lysate. Improper performance of this step is a likely source of contamination.
  6. Immunoprecipitate the protein of choice
    Wash the antibody-coupled beads carefully to eliminate any free antibody (see note). Add 400 μl of Myc-coupled beads (20% suspension, so ~80 μl bead volume) to each IP sample using a 1 ml pipette tip that has been cut off to increase the bore. Bring the total volume of each IP up to 5 ml with buffer. Incubate overnight (12-20 h) on a rocker at 4 °C.
    Note: When using any antibody-coupled bead, follow the supplier's recommended bead prewash procedure to avoid bringing along free antibody. To wash an entire bottle of 50% Sigma anti-Myc bead suspension, remove the beads from the supplier's bottle with a 1 ml pipette and follow with 2 washes of 2 ml of lysis buffer to transfer all of the beads into a 15 ml conical tube. Vortex the suspension briefly (or just mix vigorously by hand) and centrifuge for 2 min at 2,000 rpm in a tabletop centrifuge to bring the beads down. Wash the beads 3 times with 4-5 ml fresh lysis buffer each time. Finally, add lysis/IP buffer to the beads to reach a total volume of 5 ml. This amount of beads is sufficient for 12 IP's using ~400 μl of 20% suspension for each IP.

Day 2

  1. Remove the IP supernatant
    Pellet the beads in a tabletop centrifuge (3,000 rpm for S min) and remove the supernatant. Add 600 μl of lysis buffer and transfer the beads to a fresh 1.5 ml microcentrifuge tube using a 1 ml pipette tip. Repeat with 600 μl of lysis buffer to collect any residual beads.
  2. Wash the IP beads
    Between washes, spin the beads down for 1 min at 1,000 x g (3,000 rpm in an Eppendorf S417 microcentrifuge) and remove the supernatant with a small pipette tip attached to an aspirator, taking care to avoid the pellet. Perform the washes on a rocker (at RT or in the cold room) with 1 ml of the indicated solution for 5 min. Twice with lysis buffer (the first wash was done with the transfer of beads). Once with lysis buffer/500 mM NaCI. Twice with IP wash solution. Once with TE. When aspirating away the last wash, thoroughly remove the small amount of remaining TE from the beads.
    Notes: It may be useful to save the IP supernatant fraction to analyze protein content and IP efficiency.
  3. Elute the immunoprecipitate with TES (TE/1% SDS)
    Elute the immunoprecipitate from the beads with 100 μl of TE/1% SDS (PH 8.0), incubating at 65 °C for 15 min. Mix the samples briefly after 10 min. Pellet the beads for a few seconds at full speed (14,000 rpm) and transfer the eluate to a 1.5 ml tube. Add 150 μl of TE/0.67% SDS to the beads, heat for a few minutes and pellet again. Remove the supernatant and add it to the first eluate fraction. Spin the pooled eluate once more to pellet residual beads, and transfer it to a screw-cap tube, avoiding the ~10 μl left with the beads at the bottom of the tube.
  4. Reverse crosslinking
    Incubate the eluates at 65 °C over night to reverse the crosslinking.
    Notes: Screw-cap microcentrifuge tubes eliminate evaporation during the heating.

Day 3

  1. Cool the samples down at RT. Briefly spin down to collect condensation. Purify the samples using a spin column designed for small DNA fragments. Qiagen MinElute kit or PCR purification kit can be used for this step. Follow the manufacturer's instructions for using the kit.
    Note: One can vary the volume of EB as needed, keeping in mind that the DNA sequencing library construction protocol is set up for a 34 μl sample.

Notes

  1. DNA quantification
    The precipitated DNA can be quantified with NanoDrop and assayed for emichment of transcription factor bound sequences by PCR or microarray (see below). If too little DNA is purified or the DNA is not emiched for a subset of sequences, some parameters of the chromatin IP procedure can be altered, as described below.
  2. Quantitative PCR
    If you know of some sites where the protein of interest will be bound, you can use them as positive controls to assay the ChIP. This is the best way to quantitatively determine the success or failure of an experiment. See the protocol for qPCR of ChIP DNA samples.
  3. Optimizing crosslinking
    Extent of crosslinking can be adjusted by changing the time of incubation with the cross linking agent, the concentration of formaldehyde, or the temperature of crosslinking. The extent of crosslinking is critical and can depend on the individual protein. Too much crosslinking may mask epitopes, while too little will cause failure to co-IP chromatin.
  4. Assaying sonication
    Sonication should be monitored since chromatin fragments that are too large will pellet with the lysate debris. The settings described in the protocol were empirically tested by experimenters in Snyder lab. Since different sonifiers and tips may perform differently, it is strongly recommended that users adjust sonication parameters to different levels and monitor the resulting chromatin fragment size. To check DNA fragment size, take a 250 μl aliquot of the total chromatin (lysate just before the IP step) and add 250 μl of TE/1% SDS. Incubate for 6-8 h at 65 °C to reverse crosslinking, then add 20 μl of 20 mg/ml protease K and incubate for 2 h at 37 °C. Add 50 μl of 5 M LiCI, extract (3x with Phenol-Chloroform-Isopropanol, 1x with chloroform) and ethanol-precipitate the DNA (add 1 ml of ethanol, chill at -20 °C for 1 h). Resuspend the DNA in 50 μl of TE and add 2 μl of DNase-free RNase A. Incubate for 30 min at 37 °C. To resolve the DNA fragments, add DNA loading buffer to the sample. Use a loading buffer with only xylene cyanol as a marker, since it runs just above 3 kb. Pour a 1.5% agarose gel and run the gel until the marker is very well separated. The range of fragment sizes should be 100-1,000 bp, averaging 400-500.
    Input control DNA can be purified using the same procedure.
  5. Optimizing antibody amount
    The amount of antibody used for IP is another critical parameter. Preliminary IP experiments should be performed to determine the appropriate amount of antibody to be used for purification of the specific protein of interest. The amount used in this protocol is specifically tested for the myc-tagged Ste12 protein. To ensure that the crosslinking is not rendering the protein refractory to immunoprecipitation, the IP supernatant from step 11 can be analyzed by SDS-PAGE and immunoblotting. The material should be boiled in sample buffer for 20 min before running a protein gel.
  6. Cell lysis
    If necessary, it is possible to increase the efficiency of cell lysis by either increasing the number of cycles in the FastPrep machine or using rnore beads. The new FastPrep machine should give >95% lysis if used as described in the protocol.
  7. Two-step IP
    This is an alternative to use when no bead-coupled antibody is available.
    primary IP: Add the appropriate amount of free primary antibody against the protein of interest (or epitope tag) to the lysate (see Notes for determination of antibody amount to use). Incubate overnight on a rocker at 4 °C.
    secondary IP: Add 50 μl (of ~50% suspension) of protein A or G sepharose beads. Incubate on a rocker at 4 °C for 1-2 h.
    Note: This is a high-affinity binding step, and extending time is not likely to improve the IP.

Recipes

  1. TBS (1 L 10x stock)
    200 ml     1 M Tris/HCI (pH 7.6)
    300 ml     5 M NaCI
    H2O to reach 1 L
    Note: Dilute to working concentration and store in the cold room, as it is to be used cold.
  2. Lysis/IP buffer (1 L)
    50 ml 1 M Hepes/KOH (pH 7.5)
    28 ml 5 M NaCI
    2 ml 500 mM EDTA
    100 ml 10% Triton X-100
    1 g Na-deoxycholate
  3. Lysis buffer/500 mM NaCl (250 ml)
    Add NaCI to Lysis/IP buffer to bring the NaCl concentration up to 500 mM. For 250 ml final volume of Lysis buffer/500 mM NaCl, this requires 18 ml of 5 M NaCl.
  4. IP wash solution (250 ml)
    2.5 ml 1 M Tris/HCl (pH 8.0)
    12.5 ml 5 M LiCl
    6.25 ml 20% NP-40
    1.25 g Na-deoxycholate
    0.5 ml 500 mM EDTA
  5. TE/1% SDS (100 ml)
    5 ml 1 M Tris/HCl (pH 8.0)
    2 ml 500 mM EDTA
    5 ml 20% SDS
  6. TE/0.67% SDS (100 ml)
    5 ml 1 M Tris/HCl (pH 8.0)
    2 ml 500 mM EDTA
    3.35 ml 20% SDS
  7. TE (PH 8.0) (100 ml)
    5 ml 1 M Tris/HCl (pH 8.0)
    2 ml 500 mM EDTA
  8. 1 mM PMSF
    Prepare 100 mM PMSF stock solution (17.4 mg/ml) in isopropanol, and store small aliquots (0.5-1 ml) at -20 °C. Alternatively, use 100 mM PMSF.

References

  1. Aparicio, O., Geisberg, J. V. and Struhl, K. (2004). Chromatin immunoprecipitation for determining the association of proteins with specific genomic sequences in vivo. Curr Protoc Cell Biol Chapter 17: Unit 17 17.
  2. Zheng, W., Zhao, H., Mancera, E., Steinmetz, L. M. and Snyder, M. (2010). Genetic analysis of variation in transcription factor binding in yeast. Nature 464(7292): 1187-1191.

材料和试剂

  1. 500ml对数期酵母细胞培养物/ChIP(以〜0.9×10 7个细胞/ml,〜4.5×10 9个细胞/样品)
  2. 37%甲醛
  3. 2.5 H甘氨酸的H 2 O(加热灭菌的)
  4. 液氮,干冰/乙醇浴或-70℃冰箱
  5. 0.5mm氧化锆/二氧化硅珠(Bio Spec Products,目录号:11079105z)
  6. 商业蛋白酶抑制剂鸡尾酒,例如:
    Roche完全蛋白酶抑制剂混合物片剂(F.Hoffmann-La Roche,目录号:11697498001)
    Roche Complete Mini蛋白酶抑制剂混合物片剂(F.Hoffmann-La Roche,目录号:11836153001)
  7. EZview抗Myc亲和凝胶(红色珠)(Sigma-Aldrich,目录号:E6654)
  8. 用于最终DNA纯化的Minelute试剂盒(QIAGEN,目录号:28004)
  9. LiCl
  10. NaOAc
  11. 乙醇
  12. Triton X-100
  13. TE
  14. NaCl
  15. EDTA
  16. 异丙醇
  17. NP-40
  18. 脱氧胆酸钠
  19. SDS
  20. Tris缓冲盐水(TBS)(10x储备液)(见Recipes)
  21. 裂解/IP缓冲液(见配方)
  22. 裂解缓冲液/500mM NaCl(见配方)
  23. IP洗涤溶液(见配方)
  24. TE/1%SDS(100ml)(参见配方)
  25. TE/0.67%SDS(100ml)(参见配方)
  26. TE(100ml)(PH8.0)(见配方)
  27. 1mM PMSF(Fluka,目录号:93482)(参见Recipes)

设备

  1. Millipore stericup无菌真空过滤单元,500ml漏斗,0.22μm或0.45μm孔径(EMD Millipore,目录号:SCGVU05RE,SCHVU11RE)
  2. 注射器针(BD Biosciences,目录号:305155或305156)
  3. 5毫升快速盖帽管,聚丙烯(优选)或聚苯乙烯(BD Biosciences,Falcon )。
  4. 15和50ml圆锥形聚丙烯螺旋盖管(BD Biosciences,Falcon )
  5. 具有微尖或数字超声波仪S-450D(BD Biosciences)的Branson Sonifier 250,
  6. 冷藏台式离心机,例如, Beckman GS-6R,GS-15R(Beckman Coulter)或Eppendorf冷冻多功能离心机(Eppendorf?model,型号:5810R和5804R)(或简单地将普通的台式离心机放置在冷室中)
  7. 通风柜
  8. FastPrep机(FastPrep,目录号:6004500)
  9. 血细胞计数器
  10. 分光光度计

程序

第0天

  1. 设置实验。
    每个IP来自中值对数期的500ml细胞,OD 600大约为0.6,密度为〜0.9×10 7个细胞/ml。 每个IP的细胞总数为〜4.5×10 9个,每个样品的总细胞重量应为0.2-0.25g。
    注意:其他ChIP协议以10 单元格/ml或10 9 总细胞。 如果对细胞数量有疑问(例如,当处理块状酵母菌株时),使用血细胞计数器计数细胞而不使用分光光度计。
  2. 在所需条件下生长细胞
    诱导信息素应答转录的生长条件描述于Zheng等人(2010)。
  3. 用甲醛处理细胞以交联蛋白质和DNA
    在通风橱中,向细胞中加入37%甲醛至终浓度为1%(使用14ml甲醛)。保持细胞在室温(RT)15分钟,偶尔涡旋混合。有效的固定条件根据将被免疫沉淀的蛋白质而变化。最优化这个变量的最简单的方法是改变(在大多数情况下增加)固定时间
  4. 用甘氨酸猝灭交联反应 加入2.5M甘氨酸至终浓度125mM(20倍稀释,因此加入27ml)。在室温下孵育样品5分钟,偶尔混匀
  5. 收集和洗涤细胞
    通过使用0.45或0.22μm过滤器过滤收集细胞。在室温下用100ml水在过滤器上洗涤细胞两次。使用20ml水冲洗细胞从过滤器,并将其转移到50毫升聚丙烯管。重复冲洗以收集残余细胞。
    以4000rpm旋转5-25分钟以沉淀细胞并丢弃上清液。将细胞重悬在1ml水中,并将其转移到2ml螺旋盖管(用于后续裂解)。在微量离心机中旋转细胞(3分钟,最大rpm),彻底去除上清液。此时称重样品。每500ml培养物应产生0.2-0.25g细胞。向每个样品中加入1ml锆珠,以制备用于裂解步骤。将样品保存在冰上或冷冻保存。分别从这里处理实验重复。
    注意:
    1. 检查回收的细胞的总重量是一个好主意。细胞重量可以是菌株依赖性的,并且在单倍体和二倍体之间显着不同
    2. 细胞可以在此时在冰上保持几个小时或冷冻储存。将样品直接放入-70°C的冷冻箱可以很好地工作。

第1天

  1. 用蛋白酶抑制剂制备裂解/IP缓冲液
    为每个细胞样品制备6ml裂解/IP缓冲液,一些额外的用于平衡抗体珠(对于6个ChIPs为50ml)。使用罗氏完全蛋白酶抑制剂丸,将处理50毫升缓冲液。当使用平板电脑时,仍然需要添加PMSF。向每个样品管中加入0.75ml含蛋白酶抑制剂的裂解/IP缓冲液 注意:最好在使用缓冲液之前最后添加PMSF,因为它在水溶液中不稳定,pH为8时半衰期约为35分钟。
  2. 用立方锆珠溶解细胞
    在冰/水浴中进行所有操作。使用FastPrep机器,使用总共五个1分钟爆发以速度6.0(仅在需要时额外的轮次)中断细胞。每次爆裂后,将样品浸入冰水中 分钟左右,以保持他们冷。
    注意:在显微镜下检查细胞以检查有效的裂解。裂解细胞的数量应接近100%。
  3. 回收粗裂解液
    为每个样品准备一个Falcon Sml卡口管。将2 ml裂解管自上而下放在台面上。加热注射器针头在火焰中炽热,并使用它刺穿每个样品管的底部。把2毫升管约1厘米进入一个5毫米Falcon管,在那里它应该紧贴。在桌面(优选冷冻)中以1,500rpm离心1分钟,使裂解物下降到Falcon管中。向每个管中加入0.75ml冷裂解缓冲液以洗涤珠子并再次旋转。转移裂解物到15毫升锥形管(有利于超声处理),并加入2.4毫升裂解缓冲液。裂解液总体积应为〜4 ml
  4. 超声裂解液剪切染色质
    通过用装有微尖的Branson 2S0 Sonifier超声处理悬浮液来剪切染色质。在振幅6和100%占空比下使用超声波仪。每个样品超声处理5次,每次30秒。在声波处理的同时,将管保持在冰/水的小烧杯中。在声处理之间将样品在冰/水中冷却至少2分钟。还要定期(在18次超声处理后)将超声波振荡器尖端置于冰水中,以防止其过热。如果使用数字超声波S-450,每次使用15次,每次10秒,以避免过热。设置总运行时间为2分30秒,振幅50%,脉冲10秒,脉冲关1分钟。将管保持在冰/水的小烧杯中。优选地,整个过程在冷室中进行。 DNA超声处理后的平均长度应为500 bp,范围为100-1000 bp。
    注意:
    1. 应监测和调节超声处理以产生所需的平均DNA长度,如注释部分所述。
    2. 使用后清洁超声波探头。
    3. 建议的程序是将探针浸入0.1%SDS中,然后用水冲洗,用乙醇喷雾并用kimwipe组织干燥。
  5. 通过离心从裂解物中除去细胞碎片
    离心裂解物在3,000 rpm在冷藏的台式离心机在4℃下5分钟,删除上清液,并将其分成两个2毫升微量离心管。在冷却的微量离心机中以14,000rpm离心10分钟,除去上清液,将两个裂解物汇集到新鲜的15ml锥形管中。溶解产物现在可以用于IP,并且可以在此时保存等分试样用于分析总染色质和蛋白质。这通常用作输入控制。
    注意:避免携带任何聚集的碎片,保持离开沉淀,牺牲〜50μl的裂解液。此步骤的执行不当可能是污染的根源。
  6. 免疫沉淀选择的蛋白质
    仔细洗涤抗体偶联珠,以消除任何游离抗体(见注释)。加入400微升的Myc耦合珠(20%的悬浮液,所以〜80微升珠体积)到每个IP样品使用1毫升移液管尖已切断增加 孔。使用缓冲液使每个IP的总体积达到5ml。在摇床上在4℃下孵育过夜(12-20小时)。
    注意:当使用任何抗体偶联珠时,请遵循供应商推荐的珠预洗程序,以避免带来游离抗体。为了洗涤整个50%Sigma抗Myc珠悬浮液,用1ml移液管从供应商的瓶中移除珠子,然后2次洗涤2ml裂解缓冲液,将所有珠子转移到15ml锥形管中。涡旋悬浮液短暂(或只是手动混合剧烈),并在台式离心机中以2,000 rpm离心2分钟,使珠子下降。每次用4-5ml新鲜裂解缓冲液洗涤珠子3次。最后,向珠子中加入裂解/IP缓冲液以达到5ml的总体积。这个量的珠足以用于每个IP使用〜400μl的20%悬浮液的12个IP。

第2天

  1. 取出IP上清液
    在台式离心机中(3000rpm,Smin)沉淀珠子,并除去上清液。加入600μl裂解缓冲液,并使用1毫升移液器吸头将珠转移到新鲜的1.5毫升微量离心管。用600μl裂解缓冲液重复,收集任何残留的珠子。
  2. 清洗IP珠子
    在洗涤之间,将珠子以1,000xg(3,000rpm在Eppendorf S417微量离心机中)旋转1分钟,并用连接到吸气器的小吸头吸取上清液,注意避免沉淀。执行洗涤 在摇床(在RT或在冷室)中用1ml所示溶液5分钟。用裂解缓冲液两次(第一次洗涤用珠的转移完成)。一次用裂解缓冲液/500mM NaCl。用IP洗涤溶液两次。一次与TE。当吸出最后一次洗涤时,从珠粒中彻底去除少量剩余的TE 注意:保存IP上清液部分以分析蛋白质含量和IP效率可能是有用的。
  3. 用TES(TE/1%SDS)洗脱免疫沉淀物
    用100μlTE/1%SDS(PH8.0)从珠上洗脱免疫沉淀物,在65℃温育15分钟。 10分钟后短暂混合样品。在全速(14,000rpm)下将珠子球粒化几秒钟,并将洗脱液转移到1.5ml管中。加入150微升TE/0.67%SDS的珠子,加热几分钟,并再次沉淀。取出上清液,加入第一次洗脱液中。再次旋转汇集的洗脱液以沉淀残留的珠粒,并将其转移到螺旋盖管中,避免〜10μl留在管底部的珠子。
  4. 反向交联
    孵育洗脱液在65°C过夜,以扭转交联。
    注意:螺旋盖微量离心管在加热过程中消除蒸发。

第3天

  1. 在RT下冷却样品。短暂旋转收集冷凝水。使用专为小DNA片段设计的旋转柱纯化样品。 Qiagen MinElute试剂盒或PCR纯化试剂盒可用于该步骤。按照制造商的使用说明进行操作。
    注意:可以根据需要改变EB的体积,记住DNA测序文库构建方案设置为34μl样品。

笔记

  1. DNA定量
    沉淀的DNA可以用NanoDrop定量,并通过PCR或微阵列(见下文)测定转录因子结合序列的分布。如果纯化太少的DNA或DNA不显示序列的子集,则可以改变染色质IP程序的一些参数,如下所述。
  2. 定量PCR
    如果你知道一些感兴趣的蛋白质将绑定的网站,你可以使用它们作为阳性对照来测定ChIP。这是定量确定实验成功或失败的最佳方法。参见协议 用于ChIP DNA样品的qPCR
  3. 优化交联
    交联程度可以通过改变与交联剂一起孵育的时间,甲醛浓度或交联温度来调节。交联的程度是关键的,并且可以取决于单个蛋白质。过多的交联可能掩蔽表位,而太少则会导致co-IP染色质的失败
  4. 分析超声处理
    应该监测超声处理,因为太大的染色质片段会与裂解物碎片一起沉淀。协议中描述的设置由Snyder实验室的实验者进行经验性测试。由于不同的超声波仪和提示可能执行不同,强烈建议用户调整超声处理参数到不同的水平,并监控所得到的染色质片段大小。要检查DNA片段大小,取一个250微升等分的总染色质(溶胞产物在IP步骤之前),并加入250微升TE/1%SDS。孵育6-8 h在65℃反向交联,然后加入20μl的20 mg/ml蛋白酶K,并在37°C孵育2小时。加入50μl5 M LiCl,提取液(3x,用苯酚 - 氯仿 - 异丙醇,1x用氯仿),乙醇沉淀DNA(加入1 ml乙醇,-20℃冷冻1 h)。重悬在50μlTE中的DNA,加入2μl无DNA酶的核糖核酸酶A.在37℃下孵育30分钟。为了分离DNA片段,向样品中加入DNA上样缓冲液。使用加载缓冲液,只有二甲苯cyanol作为标记,因为它运行刚刚3 kb。倒入1.5%琼脂糖凝胶,运行凝胶直到标记物分离得很好。片段大小的范围应为100-1,000bp,平均值为400-500 可以使用相同的方法纯化输入对照DNA
  5. 优化抗体量
    用于IP的抗体的量是另一个关键参数。应进行初步IP实验以确定用于纯化感兴趣的特定蛋白质的抗体的适当量。该方案中使用的量特异性测试了myc标记的Ste12蛋白。为了确保交联不使蛋白质难以免疫沉淀,可以通过SDS-PAGE和免疫印迹分析来自步骤11的IP上清液。材料应在样品缓冲液中煮沸20分钟,然后运行蛋白质凝胶
  6. 细胞裂解
    如果需要,可以通过增加FastPrep机器中的循环数目或使用更少的珠子来增加细胞裂解的效率。如果按照方案中所述使用,新的FastPrep机器应该提供> 95%的裂解
  7. 两步IP
    这是当没有珠偶联抗体可用时的替代方案。
    初级IP:向裂解物中加入适当量的针对目标蛋白(或表位标签)的游离一级抗体(参见用于测定使用的抗体量的注释)。在摇床上在4℃下孵育过夜。
    二级IP:加入50μl(〜50%悬浮液)蛋白A或G琼脂糖珠。在摇床上在4°C孵育1-2小时。
    注意:这是一个高亲和力绑定步骤,延长时间不太可能改善IP。

食谱

  1. TBS(1 L 10x股票)
    200 ml     1M Tris/HCl(pH 7.6)
    300 ml     5 M NaCl
    H2O达到1L
    注意:稀释到工作浓度,储存在寒冷的房间,因为它是冷的。
  2. 裂解/IP缓冲液(1 L)
    50ml 1M Hepes/KOH(pH7.5)
    28ml 5M NaCl 2ml 500mM EDTA
    100ml 10%Triton X-100 1克脱氧胆酸钠
  3. 裂解缓冲液/500mM NaCl(250ml) 将NaCl加入到Lysis/IP缓冲液中以使NaCl浓度达到500mM。 对于250ml最终体积的裂解缓冲液/500mM NaCl,这需要18ml 5M NaCl
  4. IP洗涤溶液(250ml) 2.5ml 1M Tris/HCl(pH8.0) 12.5ml 5M LiCl
    6.25ml 20%NP-40
    1.25克去氧胆酸钠 0.5ml 500mM EDTA
  5. TE/1%SDS(100ml) 5ml 1M Tris/HCl(pH 8.0)
    2ml 500mM EDTA
    5ml 20%SDS
  6. TE/0.67%SDS(100ml) 5ml 1M Tris/HCl(pH 8.0)
    2ml 500mM EDTA
    3.35ml 20%SDS
  7. TE(PH8.0)(100ml) 5ml 1M Tris/HCl(pH 8.0)
    2ml 500mM EDTA
  8. 1mM PMSF
    准备100毫米PMSF储备液(17.4毫克/毫升)在异丙醇,并存储小等分(0.5-1毫升)在-20°C。 或者,使用100mM PMSF

参考文献

  1. Aparicio,O.,Geisberg,J.V.and Struhl,K。(2004)。 用于确定蛋白质与特定基因组关联的染色质免疫沉淀 序列。 Curr Protoc Cell Biol Chapter <
  2. Zheng,W.,Zhao,H.,Mancera,E.,Steinmetz,L.M。和Snyder,M。(2010)。 酵母中转录因子结合变异的遗传分析。 em> 464(7292):1187-1191。
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How to cite this protocol: Zheng, W. (2011). Yeast Transcription Factor Chromatin Immunoprecipitation. Bio-protocol Bio101: e92. DOI: 10.21769/BioProtoc.92; Full Text



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