广州赛诚生物ChIP核心实验技术：实验流程

相关专题

一般流程

甲醛处理细胞---收集细胞，超声破碎---加入目的蛋白的抗体，与靶蛋白-DNA复合物相互结合---加入ProteinA，结合抗体-靶蛋白-DNA复合物，并沉淀---对沉淀下来的复合物进行清洗，除去一些非特异性结合---洗脱，得到富集的靶蛋白-DNA复合物---解交联，纯化富集的DNA-片断---qPCR分析。

在qPCR分析这一块，比较传统的做法是半定量-PCR。但是现在随着荧光定量PCR 的普及，广州赛诚生物科技有限公司建议使用qPCR，更能反映实验结果。

此外，RIP其实就是用chip 的方法研究细胞内蛋白与RNA的相互结合，具体方法和ChIP差不多，只是实验过程中要注意防止RNase，最后分析的时候需要先将RNA逆转录成为cDNA; ChIP-chip其实就是ChIP富集得到的DNA-片段，拿去做芯片分析，做法在ChIP的基础上有所改变，不同的公司有不同的做法，要根据公司的要求来准备样品。

传统的ChIP实验需要3天实验时间，但广州赛诚生物科技 有限公司向您推荐的Pierce Agarose ChIP Kit(One day ChIP-qPCR)仅需1天时间。以下为这两种方法的实验流程。

一般Protocol

以下介绍的经典操作流程需要三天时间。

第一天

(一)、细胞 的甲醛交联与超声破碎。

1、取出1平皿细胞(10cm平皿)，加入243 ul 37%甲醛，使得甲醛的终浓度为1%(培养基 共有9ml)。

2、37摄氏度孵育10min。

3、终止交联：加甘氨酸至终浓度为0.125M。450 ul 2.5M甘氨酸于平皿中。混匀后，在室温下放置5min即可。

4、吸尽培养基，用冰冷的PBS 清洗细胞2次。

5、细胞刮刀收集细胞于15ml离心管 中(PBS依次为5ml，3ml和3ml)。预冷后2000rpm 5min收集细胞。

6、倒去上清。按照细胞量，加入SDS Lysis Buffer。使得细胞终浓度为每200ul含2×106个细胞。这样每100ul溶液含1×106个细胞。再加入蛋白酶抑制剂复合物。假设MCF7长满板为5×106个细胞。本次细胞长得约为80%。即为4×106个细胞。因此每管加入400ul SDS Lysis Buffer。将2管混在一起，共800ul。

7、超声破碎：VCX750，25%功率，4.5S冲击，9S间隙。共14次。

(二)、除杂及抗体哺育。

8、超声破碎结束后，10，000g 4oC离心10min。去除不溶物质。

留取300ul做实验，其余保存于-80oC。

300ul中，100ul加抗体做为实验组;100ul不加抗体做为对照组;100ul加入4ul5MNaCl(NaCl终浓度为0.2M)，65oC处理3h解交联，跑电泳，检测超声破碎的效果。

9、在100ul的超声破碎产物中，加入900ulChIPDilutionBuffer和20ul的50×PIC。

再各加入60ulProteinAAgarose/SalmonSpermDNA。4oC颠转混匀1h。

10、1h后，在4摄氏度静置10min沉淀，700rpm离心1min。

11、取上清。各留取20ul做为input。一管中加入1ul抗体，另一管中则不加抗体。4oC颠转过夜。

(三)、检验超声破碎的效果。

取100ul超声破碎后产物，加入4ul5MNaCl，65oC处理2h解交联。分出一半用酚/氯仿抽提。电泳检测超声效果。

第二天

(一)、免疫复合物的沉淀及清洗。

12、孵育过夜后，每管中加入60ulProteinAAgarose/SalmonSpermDNA。4oC颠转2h。

13、4oC静置10min后，700rpm离心1min。除去上清。

14、依次用下列溶液清洗沉淀复合物。清洗的步骤：加入溶液，在4oC颠转10min，4oC静置10min沉淀，700rpm离心1min，除去上清。

洗涤溶液：a.low salt wash buffer-one wash

b.highsalt wash buffer-one wash

c.LiCl wash buffer-one wash

d.TE buffer-two wash

15、清洗完毕后，开始洗脱。洗脱液的配方：100ul10%SDS，100ul1MNaHCO3，800ulddH2O，共1ml。

每管加入250ul洗脱buffer，室温下颠转15min，静置离心后，收集上清。重复洗涤一次。最终的洗脱液为每管500ul。

16、解交联：每管中加入20ul 5M NaCl(NaCl终浓度为0.2M)。

混匀，65oC解交联过夜。

第三天

(一)、DNA样品的回收

17、解交联结束后，每管加入1ulRNaseA(MBI)，37oC孵育1h。

18、每管加入10ul0.5MEDTA，20ul1MTris.HCl(PH6.5)，2ul10mg/ml蛋白酶K。

45oC处理2h。

19、DNA片段的回收----omega胶回收试剂盒。最终的样品溶于100ulddH2O。

(二)、qPCR分析

Pierce Agarose ChIP Kit Protocol(One day ChIP-qPCR)

该试剂盒具有以下多方面的特征，可在一天内完成经典ChIP实验流程需要三天才能完成的实验结果。

• 简单和快速的Protocl

• 高效地分离和溶解细胞核

• 简单和可重复利用的酶的消化

• 低背景高结合能力的A/ G蛋白琼脂糖树脂

• 高特异性RNA聚合酶II抗体和以GAPDH PCR引物作阳性样

• 快速和可重复利用的离心吸附柱形式

• 高回收率的DNA纯化

以下由广州赛诚生物科技有限公司为您提供该试剂盒的Protocol。

A. Experimental Design

• When designing a ChIP experiment, consider the number of immunoprecipitations desired from the chromatin sample and the number of cell culture conditions (e.g., drug-treated vs. non-treated). For accurate comparison, immunoprecipitate the same target proteins and controls from each cell culture condition.

• Performing a ChIP using the Normal Rabbit IgG is an effective negative control.

• The included anti-RNA polymerase II antibody and GAPDH primers provide a positive control for assay technique and reagent integrity. Perform a single RNA polymerase II immunoprecipitation on isolated chromatin from the control (non-treated) cell culture condition. Amplify the resulting enriched DNA with the provided GAPDH primer pair. The strength of the RNA Polymerase II IP signal should be > 50% relative to the total input control in a well-functioning assay (see Troubleshooting Section).

• If performing multiple ChIPs from a single cell culture condition, crosslinked chromatin may be prepared in bulk. Scale the reagent amounts according to the number of ChIPs desired (Table 1) and sub-divide the chromatin sample after diluting with the 1X IP Dilution Buffer. The total input control obtained from the cell culture condition is common to this set of ChIPs.

Table 1. Reagent volumes to use based on the number of ChIPs.

B. Material Preparation

• Prepare the reagents listed below once and use for all 30 ChIP reactions.

• The amounts listed below are for preparing one ChIP. If you are preparing chromatin in bulk or performing multiple ChIPs, multiply the reagent amounts by the number of ChIPs being performed (see Table 1).

C. Crosslinking and Cell Pellet Isolation

Note 1: Perform all centrifugations at room temperature.

Note 2: This procedure is for one ChIP. Multiply reagent amounts by the number of ChIPs being performed (see Table 1).

1. Culture adherent mammalian cells and treat as desired.

Optional: If you are unfamiliar with cell type being used, culture an extra dish of cells for determining cell number. Before crosslinking, trypsinize and determine the cell number from the extra dish of cells.

2. To each dish containing cell culture media, add sufficient quantity of 16% formaldehyde to obtain a final concentration of 1% formaldehyde.

Caution: Formaldehyde is a skin irritant and the fumes are toxic. Use proper personal protective, laboratory safety and disposal equipment.

3. Mix well by gently swirling the dish. Incubate at room temperature for 10 minutes in a chemical fume hood.

4. To each dish containing cell culture media and formaldehyde, add Glycine Solution (10X) to a final concentration of 1X. Mix well by gently swirling the dish. Incubate at room temperature for 5 minutes in the chemical fume hood.

5. Aspirate formaldehyde/glycine-containing media in the fume hood. Properly dispose the formaldehyde-containing waste.

6. Wash the cells twice with one media volume of ice-cold PBS, removing each wash by aspiration.

7. Add 10μL of the Halt Cocktail to 1mL of ice-cold PBS. Add the solution to the cells, and detach cells by scraping. Transfer the cell suspension to a 1.5mL microcentrifuge tube using a pipette

8. Centrifuge tubes at 3000 × g for 5 minutes.

9. Remove the PBS. Store the cell pellet(s) at -80°C, or proceed directly to Section D: Lysis and MNase Digestion.

D. Lysis and MNase Digestion

Note: For best results, empirically determine the optimal crosslinking time and Micrococcal Nuclease digestion conditions for each cell type (see Appendix A).

1. Use the crosslinked cells prepared above. If frozen, thaw cells on ice.

2. Add100μL Lysis Buffer 1 containing protease inhibitors to the cell pellet and pipette up and down to break up the pellet. Vortex the tube for 15 seconds and incubate on ice for 10 minutes.

3. Centrifuge at 9000 × g for 3 minutes and remove the supernatant.

4. Resuspend nuclei in 100μL of MNase Digestion Buffer Working Solution.

5. Add 0.25μL of Micrococcal Nuclease (ChIP Grade) (10 U/μL), vortex the tube and incubate in a 37°C water bath for 15 minutes, mixing by inversion every 5 minutes.

6. Add 10μL of MNase Stop Solution to stop the reaction, vortex briefly and incubate on ice for 5 minutes.

7. Centrifuge at 9000 × g for 5 minutes to recover the nuclei. Remove the supernatant.

8. Resuspend nuclei in 50μL of Lysis Buffer 2 containing protease/phosphatase inhibitors and incubate on ice for 15 minutes, vortexing for 15 seconds every 5 minutes.

9. Centrifuge at 9000 × g for 5 minutes and transfer the supernatant, containing the digested chromatin, to a new 1.5mL tube. Proceed to the immunoprecipitation or store samples at -80°C.

E. Immunoprecipitation

1. Transfer 5μL of the supernatant containing the digested chromatin to a 1.5mL tube and store at -20°C. This is the 10% total input sample from one ChIP.

Note: If you are preparing chromatin in bulk, this sample can be used as a common input control for all IPs from the bulk preparation.

2. Transfer the remaining 45μL of supernatant to 450μL of 1X IP Dilution Buffer.

Note: If you are preparing chromatin in bulk, unused supernatant may be stored at -80°C for later use.

3. For each IP, add 500μL diluted lysate to a plugged spin column and add primary antibody. The following amounts of antibody are recommended when using 2 ×106 HeLa cells/IP.

Positive control IP (included): add 10μL Anti-RNA Polymerase II Antibody

Negative control IP (included): add 1 to 2μL of Normal Rabbit IgG

Target-specific IP(s): The typical concentration is 1-10μg antibody for each IP; however, titration of antibody concentration to obtain the best signal-to-noise ratio is required.

4. Incubate IP reactions for 2 hours to overnight at 4°C on a rocking platform.

Note: For low-abundant proteins, an overnight incubation greatly increases signal.

5. Cap and vortex the tube of ChIP Grade Protein A/G Plus Agarose to obtain a uniform suspension. Using a wide-bore or cut pipette tip, add 20μL of the agarose resin to each IP and incubate for 1 hour at 4°C on a rocking platform.

6. After resin incubation, remove and set aside the column plug. Place the column into a 2mL collection tube.

7. Centrifuge tube at 3000 × g for 30 seconds. Remove column from the collection tube, discard the column flow-through and set aside the collection tube.

8. Re-insert the same column plug, apply 0.5mL of IP Wash Buffer 1, cap the column and incubate at 4°C for 5 minutes on a rocking platform.

9. Remove the column plug and set aside. Place the column into the same collection tube.

10. Centrifuge at 3000 × g for 30 seconds. Remove column from the collection tube, discard the column flow-through and set aside the collection tube.

11. Repeat steps 8-10 twice with IP Wash Buffer 2.

12. Repeat steps 8-10 once with IP Wash Buffer 3.

13. To remove residual Wash Buffer, insert column into the collection tube and centrifuge at 3000 × g for 1 minute.

F. IP Elution

1. Re-fit column plug, add 150μL 1X IP Elution Buffer to the washed resin, cap the column, and incubate at 65°C for 30 minutes with shaking. If a thermomixer is unavailable, incubate the column in a heat block set a 65°C for 40 minutes. Re-suspend the beads by flicking the tube every 10 minutes.

2. During the elution step, prepare a 1.5mL microcentrifuge tube for each IP containing 6μL of 5M NaCl and 2μL of 20mg/mL Proteinase K.

3. Thaw 10% total input sample(s) and add 150μL of IP Elution buffer, 6μL of 5M NaCl and 2μL of 20mg/mL Proteinase K. Place tubes at room temperature until the next step.

4. Following the 65°C incubation, remove the column from the heat block, open the cap, remove the plug, and place the column in the previously prepared 1.5mL collection tube containing the NaCl and Proteinase K, close the column cap and centrifuge at 6000 × g for 1 minute.

Note: After incubation, it is essential to open the column cap before plug removal to equalize the pressure within the column. Failure to open the cap before plug removal will result in sample loss.

5. Discard the columns. Cap the 1.5mL centrifuge tubes, vortex all IP and total-input samples and place in a 65°C heat block for 1.5 hours.

G. DNA Recovery

1. To each eluted IP and total input sample, add 750μL of DNA Binding Buffer.

2. Pipette 500μL of each sample into a DNA Clean-Up Column inserted into a 2mL collection tube. Centrifuge the columns at 10,000 × g for 1 minute and discard the flow-through.

3. Pipette the remaining sample into the same DNA Clean-Up Column. Centrifuge the columns at 10,000 × g for 1 minute and discard the flow-through.

4. Place the column back into the collection tube and add 750μL of DNA Column Wash Buffer. Centrifuge the columns at 10,000 × g for 1 minute and discard the flow-through.

5. Place the column back in the empty collection tube and centrifuge the columns at 10,000 × g for 2 minutes.

6. Place the column in a new 1.5mL centrifuge tube and pipette 50μL of DNA Column Elution Solution directly into the center of each column.

7. Centrifuge the column at 10,000 × g for 1 minute and discard the column. The resulting solution is the purified DNA. Proceed to PCR or QPCR detection (see Appendix B).

Appendix A: Optimization of micrococcal nuclease digestion

1. Prepare crosslinked cell pellet from 107 cells as described in Section C.

2. Follow the lysis procedure in Section D, Steps 1-4; however, use 0.5mL of the Lysis Buffer 1, Membrane Extraction Buffer and MNase Digestion Buffer Working Solution.

3. Transfer 100μL of the resuspended nuclei into five tubes (2 × 106 nuclei/tube), labeling the tubes 0, 1, 2, 4 and 6.

4. Add 2μL of the Micrococcal Nuclease (ChIP Grade) to 18μL of MNase Digestion Buffer Working Solution (1:10 dilution).

5. Add 0, 1, 2, 4 and 6μL of the diluted Micrococcal Nuclease to the corresponding tubes of nuclei and incubate for 15 minutes in a 37°C water bath mixing by inversion every 5 minutes.

6. Add 20μL MNase Stop Solution to each tube and incubate on ice for 5 minutes.

7. Centrifuge at 9000 × g for 5 minutes to recover the nuclei and remove the supernatant.

8. Resuspend nuclei in 50μL of Nuclear Extraction Buffer and incubate on ice for 15 minutes, vortexing for 15 seconds every 5 minutes.

9. Centrifuge at 9000 × g for 5 minutes.

10. Prepare five 1.5mL microcentrifuge tubes labeled 0, 2, 4 and 6 each containing 6.6μL Nuclease Free Water, 2.4μL of 5M NaCl, and 1μL of Proteinase K (20mg/mL).

11. Transfer 50μL of each nuclear supernatant, containing digested chromatin, to the corresponding tube.

12. Vortex each tube for 10 seconds and incubate the tubes at 65°C in a heat block for 1.5 hours.

13. Analyze 15-25μL of each sample and DNA size marker by agarose gel electrophoresis. Optimal digestion should yield fragments from 200 to 1000 base pairs with a more intense ladder of bands occurring at approximately 160, 320, and 480 base pairs, which corresponds to the 1, 2 and 3 nucleosome units.

Appendix B: Real-time PCR analysis using positive control primers

Recommendations:

• Use filter tips, gloves, and nuclease-free reagents to prevent contamination.

• Using hot-start Taq polymerase and validated PCR master mix greatly reduces PCR artifacts and increases reaction efficiency.

• The Positive Control Primers included are designed to amplify a region of the human GAPDH promoter close to the transcription start site. The primers are validated for detecting RNA polymerase II binding to the GAPDH promoter in human-derived cells and tissue. Detection of other Protein:DNA interactions using these primers is not validated.

• When designing primers, using reputable primer design software greatly increases successful PCR detection.

• Follow the manufacturer’s recommendations when programming the thermocycler and collecting real-time data.

• Use a standard curve of serially diluted genomic DNA to evaluate PCR efficiency and linear amplification.

• Amplify each sample in triplicate to control for pipetting error.

Recommended reaction conditions for quantitative real-time PCR (qPCR) with the GAPDH control:

Thermocycler Amplification Settings:

Step 1: 95°C for 15 minutes.

Step 2: 95°C for 15 seconds.

Step 3: 62°C for 1 minute (collect real-time data).

Step 4: Repeat Steps 2 to 3 for 40 cycles.

标签: 广州赛诚生物 实验流程 ChIP实验技术

作者：广州赛诚生物