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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, 6585 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.       每次ChIP500ml 对数期生长的酵母细胞 (-0.9x107 cells/ml, -4.5x109 cells per sample)

2.       37% 甲醛

3.       2.5M 甘氨酸,水溶解 (加热消毒)

4.       液氮, 干冰/乙醇或 -70°C 冷冻

5.       微孔Stericup 无菌真空过滤装置, 500ml 漏斗, 孔径为0.22um or 0.45um (cat# SCGVU05RE, SCHVU11RE)

6.       0.5mmZirconia/Silica beads (BioSpec Products, Inc., catalog #11079105z)

7.       注射器及针头 (Becton Dickinson #305155 or 305156)

8.       TBS (tris-buffered saline, 20mM Tris/HCI, 150mMNaCI)

9.       1mM PMSF. 准备 100mM PMSF 原液 (17.4mg/ml) 异丙醇溶解,小份分装 (-0.5-1ml) -20°C储存。 或者选择 100mM PMSF Fluka, catalog #93482

10.   蛋白酶抑制剂cocktail, 例如 Roche Complete protease inhibitor cocktail tablets, Roche catalog #11697498001, Roche Complete Mini protease inhibitor cocktail tablets, Roche #11836153001

11.   Lysis/IP buffer (50mM Hepes/KOH, 140mMNaCI, 1mMEDTA, 1 % Triton X-100, 0.1% Na-deoxycholate)

12.   Lysis buffer /500mM NaCl

13.   IP wash solution (10mM Tris/HCl, 0.25M LiCI, 0.5% NP-40, 0.5% Na-deoxycholate, 1mM EDTA)

14.   TE/1% SDS (50mM Tris/HCI, 10mM EDTA, 1%SDS)

15.   TE/0.67% SDS (50mM Tris/HCI, 10mM EDTA, 0.67% SDS)

16.   TE pH 8.0 (50mM Tris/HCl, 10mM EDTA)

17.   Sigma EZview anti-Myc affinity gel (red colored beads), catalog #E6654

18.   Falcon 5ml snap-cap tubes, polypropylene (preferred) or polystyrene

19.   15 ml 50ml有螺旋盖的聚丙烯锥形管

20.   5M LiCl in H20

21.   3M NaOAc in H20

22.   100% 乙醇

23.   Minelute kit for final DNA purification, Qiagen #28004

 

仪器设备

 

1.       Branson Sonifier 250 with microtip or Digital Sonifier S-450D

2.       台式冷冻离心机,比如Beckman GS-6R, GS-15R, or EppendorfTM Refrigerated Multipurpose Centrifuge Model 5810R and Model 5804R. (或在冷藏室用普通台式离心机)

3.       通风

4.       FastPrep Machine (cat# 6004500)

 

步骤

 

0:

1.       设置实验

每次ChIP需要500ml对数中期生长的细胞,其OD6000.6,密度为0.9xI07 cells/ml,总细胞数目约4.5x109,每次样品的总细胞重量应该为0.2-0.25g。注意,其他的 ChIP 方法 指定 100ml 细胞溶液约 107 cells/ml 109 细胞。 如果对细胞数目存在疑义 (例如当存在块状酵母菌株时),用血球计数仪来替代分光光度计计算细胞数目。

2.       根据所需条件培养细胞

诱导费洛蒙反应转录的生长条件 在第2篇参考文献中有介绍。

3.       用甲醛处理细胞交联蛋白质和DNA

在通风橱中,向细胞溶液中加入37%甲醛至终浓度为 1 % ( 14ml 甲醛).室温条件下保持细胞交联15分钟,不时旋转混匀。 具体有效的固定条件取决于将要被沉淀的蛋白质。 最简单的优化方式是改变固定时间。

4.       甘氨酸终止交联反应

加入2.5M甘氨酸至体系终浓度为125mM (20倍稀释故加27ml)以终止交联反应。室温下孵育5分钟,不时混匀。

5.       收集并清洗细胞

 0.45 or 0.22uM 的过滤器过滤收集细胞,室温下用100ml水清洗两次滤膜上的细胞,最后用20ml水将滤膜上的细胞冲洗至一50ml聚丙烯管。重复冲洗收集残留细胞。

4000rpm离心 5-25分钟,收集沉淀细胞,弃掉上清。用1ml水重悬细胞并转移至2ml 离心管。用微型离心机旋转沉降细胞 (3 分钟至高速),彻底去除上清。此时称量样品的重量,每500ml培养的细胞应收集约重0.2-0.25g 加入1ml zirconium beads 至每个样品,为后面的裂解做准备。样品要一直保持在冰上或冻存。

注:1)检测下收集细胞的总重量是很有必要的,细胞的重量依赖并有分别于其倍型。2)此时的细胞可以在冰上保存数小时 或冰冻保存,将样品直接放至-70冰箱即可。

第一天

6.       准备 lysis/IP buffer(加蛋白酶抑制剂)

每一份样品准备6ml of lysis/IP buffer及多量的一些以便后面用来平衡抗体 beads (50ml 可以用做6 ChIPs)50mlbuff里加入一片罗氏的蛋白酶抑制剂药品。 除了药品,仍需加PMSF。加 没样片管里加0.75ml 蛋白酶抑制剂处理过的 IP/lysis buffer 

注意:PMSF要现用现加,因为它的水溶液不稳定,PH8时半衰期为35分钟。

7.       cubic zirconium beads裂解细胞

所有操作需要在冰上进行。FastPrep仪破碎细胞 5次,1-分钟,速度为6 (需要的时候可以增加次数)。每次爆破结束,将样品浸到冰水1分钟左右保持它们的低温。

注意: 显微镜下检测细胞裂解的效率,裂解的细胞应接近于100%

8.       回收粗糙的裂解液

给每一个样品准备一个小的带螺帽的离心管 Place the 2ml lysis tube top-down on the benchtop. 用火将注射器针头烧红,然后用它将每个样品管的底部刺穿。将2ml 管放入5ml 离心管约1cm呈紧贴状态的一个位置。 台式离心机 (最后是低温的) 1500rpm离心 1 分钟,将裂解液离心至离心管。每个样品管中加入 0.75ml预冷的lysis buffer再洗一遍 beads离心。将得到的裂解液转移15ml 锥形管 (利于超声)然后再加入2.4ml lysis buffer,终体积应该约为~4ml

9.       超声裂解液以剪切染色质

 Branson 2S0 Sonifier fitted 的微探针来超声菌体以剪切染色质。索尼法伊厄()声波降解器的振幅为6 100% 占空比。每个样品超声5次每次30秒。超声的时候保持离心管在冰上。超声的过程在冰水中至少2分钟冷却样品,同时定时的冷却超声探针防止温度过高。 如果使用的是 Digital Sonifier S-450,超声 15次每次10秒钟防止过热 。设置总运行时间为230 秒, 振幅50%,脉冲10 秒, 脉冲关闭1分钟。将样品管保持在冰上。整个过程最好在冷室中进行。超声破碎之后的DNA 的平均长度约为500bp,范围为100-1000bp

注意:对超声的效果应该做一个检测确保使最终得到的DNA的平均长度符合要求。使用后要对超生的探针清洗。建议依次将探头浸入0.1 % SDS、水、 乙醇最后擦干。

10.   离心去除裂解液中的细胞碎片

将裂解液用台式冷冻离心机43000rpm离心5分钟,将上清吸出平均分成两份至2ml离心管。14,000rpm低温离心10 分钟,转移上清至一15ml 锥形管。现在的裂解液即可用来做IP,可在此时保存等份裂解液,以便作为input对照用来对总的染色质及蛋白质进行分析。

注意:移液是不要碰到底层以避免汲取细胞碎片,丢弃最后约 ~50ul的裂解液。这一步骤操作不当就可能是污染的来源。

11.   免疫沉淀目的蛋白

小心地预洗 antibody-coupled beads以消除游离抗体。用剪断头1ml枪头吸取 400ul Myc-coupled beads (20% 悬浮液,所以加~80ul bead) 至每一个 IP样品。用缓冲液将每一个IP补至5ml4,振荡孵育过夜(12-20 hours) 

注意:用任何antibody-coupled bead时,应遵循供应商的建议预洗bead避免引入游离抗体。洗脱一整瓶的50% Sigma anti-Myc bead 悬浮液,用1ml枪头将beads从供应商的瓶子中移出,后用2mllysis buffer清洗两次,确保将所有的beads转移至一15ml 锥形瓶。短暂的斡旋混匀(或只是用手使劲混匀) ,然后用台式离心机以2000rpm离心2分钟收集beads 。每次用4-5ml新的lysis bufferbeads3次。最后加 lysis/IP buffer使beads的终体积为5ml。这些beads12 IP的用量 ~每次用400ul of 20% beads悬浮液 

第二天

12.   去除IP 上清

台式离心机 (3000rpm  S分钟) 离心bead,去除上清。加入 600ullysis buffer ,用1ml枪头将beads 转移至一新的1.5ml 离心管。加入600ullysis buffer 重复操作一次以回收残留的beads

13.   洗脱IP beads.

在每次洗脱之间,以1000g 的转速离心beads 1分钟 (3000rpm  Eppendorf S417 微型离心机) 用小枪头尽量去除上清,小心操作避免沉淀。洗脱的时候加1ml洗脱液(室温或冷室)摇动离心管5分钟。洗脱顺序为两次 lysis buffer 、一次 lysis buffer/500mM NaCI、两次 IP wash solution、一次TE。最后一步洗脱时,彻底去除beads剩余的少量TE

注意:最后保留 IP 上清以 分析蛋白质含量及IP 效率

14.   TES (TE/1% SDS)洗脱免疫沉淀

100ul TE/1% SDS (PH 8.0)65孵育15 minutes来洗脱beads上的免疫沉淀,10分钟后搅拌样品。14,000rpm离心几秒,转移洗出液至1.5ml管中。再加入150ul TE/0.67% SDS  beads,加热几分钟再沉淀。吸上清合并至第一次的洗出液。再次旋转汇集残余beads的洗脱液转移至有螺旋管的管,避免管底部约10ul的残留。

15.   解交联

 65°C 孵育洗涤液过夜以解交联。

注意:使用旋盖离心管 可以消除加热过程中的蒸发。

第三天

16.   将样品降至室温。轻甩收集冷凝的样品。利用小片段DNA回收的柱子纯化样品。Qiagen MinElute kit PCR purification kit 可以用在此步骤。按照试剂盒的只是说明操作。

注意:可以根据需要调整EB的量,谨记构建DNA 测序文库 需要34ul的样本。

 

注意事项

 

1.       DNA 的量化

沉淀得到的DNA可以通过NanpDrop检测浓度,通过PCR或芯片进一步检测转录因子结合序列。如果纯化DNA太少或者不纯 ChIP过程的一些参数可做更改,下面会有讲述。

2.       定量PCR

如果你知道一些目的蛋白的结合位点,你可以利用它们作为ChIP实验的正对照。这是最好的方法来确定你实验的成败与否。参阅 qPCRChIP DNA样品的方法。.

3.       优化交联

交联程度可以通过改变与交联剂的孵化时间、甲醛的浓度或交联温度来改变。交联的程度至关重要,具体程度依赖于具体蛋白而定。过度交联可能掩盖表位,而交联太轻则可能导致共沉淀的失败。

4.       超声质量测定

应该检测超声的质量,染色质片段太大时将于裂解液的碎片共沉淀。不同的超声仪与探头的工作效率不同,所以每个人操作的时候应根据自己的事业条件调整超声的参数,是染色质的片段达到要求。取250ul样品来检测DNA片段的大小 ( IP之前的裂解液) ,加入 250ul TE/1% SDS65孵育6-8 小时解交联,然后加入 20ul of 20mg/ml protease K 37孵育2h,加50ul of 5M LiC 抽提(3x-氯仿-异丙醇, 1x 氯仿) 乙醇沉淀 DNA (add 1ml乙醇, -20°C放置1h)50ul of TE重悬DNA,加入2ul DNase-free RNase A. 37°C孵育30分钟。加入DNA loading buffer至样品中, xylene cyanol 作为 marker 1.5%的琼脂糖凝胶,跑胶至marker分离的比较开。片段的范围应为100-1000bp, 集中在 400-500

Input对照的 DNA 可以按相同的操作步骤纯化。

5.       优化抗体量

用于实验的抗体量是另一个重要的参数。初步的免疫沉淀实验应该确定一个合适的抗体量能纯化到特异的目的蛋白。本篇中抗体的量是针对于检测 myc标记的 Ste12蛋白的。为了确保交联的成功, 步骤11 的上清可以通过SDS-PAGE及免疫印记分析。材料应在样品缓冲液里煮过20分钟后再上样跑蛋白胶。

6.       细胞裂解

必要的话,可以通过两种方法来增加细胞裂解的效率。通过增加FastPrep仪的循环数或用更多的rnore beads。利用本篇描述的条件,新FastPrep 裂解率>95%

7.       两步 IP

下面是一种没有bead-coupled antibody时的替代方法。

初步 IP: 加入目的蛋白适量的游离初级抗体(或附加表位)至裂解液中(参阅抗体的测定及使用)4°C振荡孵育过夜。

二步 IP:加入 50uI (~50% 悬浮液) protein A or G sepharose beads 4°C 振荡孵育 1-2 小时。

注意: 此步高亲和力结合的步骤,增加时间不太可能改善IP

 

配方

 

1.       TBS (1L 10x 原液):

200ml     1M Tris/HCI, pH 7.6

300ml     5M NaCI

H2O定容至1L

注意: 稀释至工作浓度使用,并应贮存在冷室,因为需要低温使用。

2.       Lysis /IP 缓冲液 1 L:

50ml 1M HepeslKOH, pH 7.5

28ml 5MNaCI

2ml   500mM EDTA

100ml      10% Triton X-100

1g     Na-deoxycholate

3.       Lysis buffer/500mM NaCl 250ml:

 NaCI Lysis/IP buffer NaCI 终浓度为500mM250ml体积的 Lysis buffer/500mM NaCI,需要5M NaCl的体积为18ml

4.       IP wash solution 250ml:

2.5ml       1M Tris/HCl, pH 8.0

12.5ml            5M LiCI

6.25ml            20% NP-40

1.25g    Na-deoxycholate

0.5mI                     500mM EDTA

5.       TE/1% SDS 100ml:

5ml   1M Tris/HCI, pH 8.0

2ml   500mM EDTA

5ml   20% SDS

6.       TE/0.67% SDS 100ml:

5ml 1M Tris/HCI, pH 8.0

2ml 500mM EDT A

3.35ml     20% SDS

7.       TE (PH 8.0) 100ml:

5ml 1M Tris/HCI, pH 8.0

2ml 500mM EDTA

 

参考

 

1.        Aparicio O., Geisberg J.V., 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 7. 

2.        Zheng W., Zhao H., Mancera E., Steinmetz L.M., Snyder M. (2010). Genetic analysis of variation in transcription factor binding in yeast. Nature 464(7292): 1187-91. 

 

 

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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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