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Chromatin Immunoprecipitation (ChIP), Streptavidin and ATP-agarose Mediated Pull-down Analyses
染色质免疫共沉淀、链酶亲和素和琼脂糖三磷酸腺苷介导的Pull-down检测技术   

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Abstract

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) induces expression of both viral and cellular genes in virus infected B cells by mimicking activated Notch receptors (Notch-IC) that mediate transcription activation through binding to the repressing domain of the recombining binding protein suppressor of hairless (RBP-Jκ). In general, chromatin immunoprecipitation (ChIP) assays, electrophoresis mobility shift assays (EMSA), streptavidin-agarose mediated DNA pull-down assays, together with cell-based transcription reporter assays were conducted to verify whether the query protein is involved in EBNA2-dependent transcription. The ATP-bound state of nuclear chaperone nucleophosmin (NPM1) has been implicated in pleiotropic biological processes. An ATP-agarose-mediated pull-down protocol was developed to monitor the formation of the pre-initiation complex that is induced by ATP-bound NPM1. According to EBNA2 and Notch-IC have been shown to be partially interchangeable with respect to activation of target genes in B cell lines, it is conceivable that EBNA2 is a biological equivalent of an activated Notch IC.

Part I:   ATP Depletion

Materials and Reagents

  1. Glucose (-) RPMI 1640 (Life Technologies, catalog number: 11879020 )
  2. 10% Fetal Calf Serum (Life Technologies, Gibco®)
  3. Penicillin/streptomycin (Life Technologies, catalog number: 15140-122 )
  4. Glutamine (Life Technologies, catalog number: 25030-081 )
  5. Deoxyglucose (Sigma-Aldrich, catalog number: D6134 )
  6. Antimycin A (Sigma-Aldrich, catalog number: A8674 )
  7. RPMI 1640 (Life Technologies, catalog number: 31800089 )
  8. ATP depletion medium (see Recipes)
  9. Culture medium for IB4 LCL (see Recipes)

Equipment

  1. CO2 incubator (Thermo Fisher Scientific Forma CO2 incubator)
  2. Centrifuge (Eppendorf, model: 5810R )

Procedure

  1. Pellet 5 x 106 of lymphoblastoid cells and replenish with 5 ml of ATP-depletion medium.
  2. Incubate cells at CO2 incubator (37 °C) for two hours.
  3. Collect cells by spin at 1,050 rpm (220 x g; Eppendorf 5810R) for 5 min.
  4. Wash cells twice with 1x PBS and save for further experimental purpose.

Recipes

  1. ATP depletion medium
    Glucose (-) RPMI 1640 was supplemented with:
    10% Fetal Calf Serum
    100 U/ml Penicillin/100 μg/ml streptomycin
    2 mM Glutamine
    2 mM deoxyglucose
    300 nM antimycin A
  2. Culture medium for IB4 LCL
    RPMI 1640 was supplemented with:
    10% Fetal Calf Serum
    100 U/ml Penicillin/100 μg/ml streptomycin
    2 mM Glutamine

References

  1. Liu, C. D., Chen, Y. L., Min, Y. L., Zhao, B., Cheng, C. P., Kang, M. S., Chiu, S. J., Kieff, E. and Peng, C. W. (2012). The nuclear chaperone nucleophosmin escorts an Epstein-Barr Virus nuclear antigen to establish transcriptional cascades for latent infection in human B cells. PLoS Pathog 8(12): e1003084.


Part II:   Streptavidin-agarose Mediated DNA Pull-down Assay

Materials and Reagents

  1. DNA template (In this case: LMP1 promoter reporter plasmid)
    Note: Transcription of LMP1 promoter is essential for EBV latent infection.
  2. Biotin-labeled primers (Vita Genomics)
  3. IB4 lymphoblastoid cells (LCL)
  4. DNA elution column (Geneaid Biotech)
  5. Streptavidin-conjugated agarose (Life Technologies, catalog number: SA100-04 )
  6. 2x SDS sample buffer
  7. NP-40 (Sigma-Aldrich, catalog number: I8896 )
  8. Pepstatin A (Sigma-Aldrich, catalog number: P5318 )
  9. Aprotinin (Sigma-Aldrich, catalog number: A1153 )
  10. Leupeptin (Sigma-Aldrich, catalog number: L2884 )
  11. Phenylmethanesulfonyl fluoride (Sigma-Aldrich, catalog number: P7626 )
  12. Lysis buffer (see Recipes)
  13. Protease inhibitors (see Recipes)

Equipment

  1. PCR product elution column (Geneaid Biotech)
  2. Centrifuge (Eppendorf, model: 5810R )
  3. Centrifuge (Eppendorf, model: 5415R )
  4. Rocker (Tube rotator)
  5. PCR machine (MJ Research, model: PTC 200 Thermal Cycler )
  6. Gel electrophoresis equipment (Embi Tec, model: RunOneTM Electrophoresis System )
  7. Western blot equipment (Bio-Rad, model: Mini-protean 3 )

Procedure

  1. Amplify the biotin or non-biotin labeled DNA fragments of the query promoter in a 100 μl reaction volume by PCR.
  2. Elute PCR product by passing through an elution column with 100 μl elution buffer and take 5 μl DNA (5% input DNA) for performing gel electrophoresis.
  3. Take 1 x 107 lymphoblastoid cells to be lysed with 1 ml of lysis buffer, put the reaction tubes on a rocker and incubate at 4 °C for 1 h. Collect supernatant by spining at 13,200 rpm (16,100 x g; Eppendorf 5415R), 4 °C.
  4. Add desired biotin-labelled or non-biotin-labelled DNA to supernatant and put tubes onto the rocker and incubate at 4 °C for another hour.
  5. Add 40 μl streptavidin-conjugated agarose to each tube and put the tubes onto the rocker again, and wait for 30 min to allow the affinity binding of streptavidin with biotin to complete.
  6. Harvest biotin-labelled DNA by spining at 1,050 rpm (220 x g; Eppendorf 5810R), 4 °C for 5 min.
  7. Wash the collected streptavidin-conjugated agarose beads with 500 μl lysis buffer five times and add 40 μl 2x SDS sample buffer to each sample and use them for western blot analysis.

Recipes

  1. Lysis buffer (for 1 L)
    1% NP-40
    2 mM EDTA
    10% Glycerol
    50 mM Tris-HCl
    150 mM NaCl
  2. Protease inhibitors
    1 μg/ml Pepstatin A
    1 μg/ml Aprotinin
    1 μg/ml Leupeptin
    0.5 mM Phenylmethanesulfonyl fluoride
    *Lysis buffer is supplemented with the indicated protease inhibitors

References

  1. Liu, C. D., Chen, Y. L., Min, Y. L., Zhao, B., Cheng, C. P., Kang, M. S., Chiu, S. J., Kieff, E. and Peng, C. W. (2012). The nuclear chaperone nucleophosmin escorts an Epstein-Barr Virus nuclear antigen to establish transcriptional cascades for latent infection in human B cells. PLoS Pathog 8(12): e1003084.


Part III:   ATP-agarose Mediated Pull-down Analyses

Materials and Reagents

  1. IB4 LCL cells
  2. Adenosine 5′-triphosphate-Agarose (Sigma-Aldrich, catalog number: A2767-1 ml )
  3. 2x SDS sample buffer
  4. Glucose (-) RPMI 1640 (Life Technologies, catalog number: 11879020 )
  5. Fetal Calf Serum (Life Technologies, Gibco®)
  6. Penicillin/streptomycin (Life Technologies, catalog number: 15140-122 )
  7. Deoxyglucose (Sigma-Aldrich, catalog number: D6134 )
  8. Antimycin A (Sigma-Aldrich, catalog number: A8674 )
  9. RPMI 1640 (Life Technologies, catalog number: 31800089 )
  10. Glutamine (Life Technologies, catalog number: 25030-081 )
  11. Pepstatin A (Sigma-Aldrich, catalog number: P5318 )
  12. Aprotinin (Sigma-Aldrich, catalog number: A1153 )
  13. Leupeptin (Sigma-Aldrich, catalog number: L2884 )
  14. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  15. ATP-depletion culture medium (see Recipes)
  16. Lysis buffer (see Recipes)
  17. Protease inhibitor cocktail (see Recipes)
  18. Culture medium for IB4 LCL (see Recipes)

Equipment

  1. CO2 incubator
  2. Centrifuge (Eppendorf, model: 5415D )
  3. Centrifuge (Eppendorf, model: 5810R )
  4. Centrifuge (Eppendorf, model: 5415R )
  5. Rocker
  6. Western blot equipment (Bio-Rad Laboratories)

Procedure

  1. Collect 1 x 107 of IB4 LCL cells by spin at 1,050 rpm (220 x g; Eppendorf 5810R) and replenish with 5 ml of ATP-depletion culture medium and incubate at CO2 incubator (37 °C) for 2 h.
  2. Harvest cells and wash them twice with 1x PBS.
  3. Lyse cells in 0.5 ml of lysis buffer and put the reaction tubes on a rocker and incubate at 4 °C for 1 h. Collect supernatant (cellular proteins) by spining at 13,200 rpm (16,100 x g; Eppendorf 5415R), 4 °C for 10 min.
  4. Add 50 μl ATP-agarose to pull down cellular protein by sitting on a rocker and incubate at 4 °C for 4 hours.
  5. Collect ATP-agarose beads by spining at 5,000 rpm (2,300 x g; Eppendorf 5415R) for 1 min and wash beads with lysis buffer five times.
  6. Add 40 μl 2x SDS sample buffer to each collected pull-down sample.
  7. Identify the ATP-bound proteins by western blot analysis.

Recipes

  1. Lysis buffer (for 1 L)
    1% NP-40
    2 mM EDTA
    10% Glycerol
    50 mM Tris-HCl
    150 mM NaCl
  2. Protease inhibitors
    1 μg/ml Pepstatin A
    1 μg/ml Aprotinin
    1 μg/ml Leupeptin
    0.5 mM Phenylmethanesulfonyl fluoride
    *Lysis buffer is supplemented with the indicated protease inhibitors
  3. ATP-depletion culture medium
    Glucose (-) RPMI 1640 was supplemented with:
    10% Fetal Calf Serum
    100 U/ml Penicillin/100 μg/ml streptomycin
    2 mM Glutamine
    2 mM deoxyglucose
    300 nM antimycin A
  4. Culture medium for IB4 LCL
    RPMI 1640 was supplemented with:
    10% Fetal Calf Serum
    100 U/ml Penicillin/100 μg/ml streptomycin
    2 mM Glutamine

References

  1. Liu, C. D., Chen, Y. L., Min, Y. L., Zhao, B., Cheng, C. P., Kang, M. S., Chiu, S. J., Kieff, E. and Peng, C. W. (2012). The nuclear chaperone nucleophosmin escorts an Epstein-Barr Virus nuclear antigen to establish transcriptional cascades for latent infection in human B cells. PLoS Pathog 8(12): e1003084.


Part IV:   Chromatin Immunoprecipitation (ChIP) Assay

Materials and Reagents

  1. ATP-depleted Lymphoblastoid cells
  2. IB4 LCL cells (Lymphoblastoid cell lines)
  3. 36.5% Formadehyde (Sigma-Aldrich, catalog number: F8775 )
  4. Glycerol (Sigma-Aldrich, catalog number: F8775 )
  5. EDTA (Sigma-Aldrich, catalog number: EDS )
  6. Chromatin Shearing Kit (Active Motif)
  7. DNA nuclease (Active Motif)
  8. Enzyme digestion buffer (Active Motif)
  9. Protein A/G agarose (EMD Millipore, catalog number: IP10-10ML )
  10. Primary antibodies (Ab1, Ab2 and H3-Ac)
    In this case:
    Ab1: EBV nuclear antigen 2 specific antibody (e.g. Abcam, catalog number: ab49498 )
    Ab2: Nucleophosmin antibody (5E3) (Santa Cruz, catalog number: sc-53926 )
    Ab3: H3-acetylated antibody (H3-Ac) (EMD Millipore, catalog number: 06599 )
  11. IgG (Dako, catalog number: A0424 )
  12. TE buffer
  13. 1 M Tris-HCl
  14. Proteinase K (Life Technologies, catalog number: 25530049 )
  15. Glucose (-) RPMI 1640 (Life Technologies, catalog number: 11879020 )
  16. RPMI 1640 (Life Technologies, catalog number: 31800089 )
  17. Fetal Calf Serum (Life Technologies, Gibco®)
  18. Penicillin/streptomycin (Life Technologies, catalog number: 15140-122 )
  19. Glutamine (Life Technologies, catalog number: 25030-081 )
  20. Deoxyglucose (Sigma-Aldrich, catalog number: D6134 )
  21. Antimycin A (Sigma-Aldrich, catalog number: A8674 )
  22. ChIP lysis buffer (see Recipes)
  23. 10x Glycine (see Recipes)
  24. ChIP dilution buffer (see Recipes)
  25. Low Salt Immune Complex Wash Buffer (see Recipes)
  26. High Salt Immune Complex Wash Buffer (see Recipes)
  27. LiCl Immune Complex Wash Buffer (see Recipes)
  28. Elution buffer (see Recipes)
  29. ATP depletion medium (see Recipes)
  30. Culture medium for IB4 LCL (see Recipes)
  31. Enzyme solution (see Recipes)
  32. Protease inhibitors (see Recipes)

Equipment

  1. DNA spin column (Geneaid Biotech)
  2. Douncer apparatus (Tissue grinder) (Thomas scientific)
  3. Centrifuge (Eppendorf, model: 5810R )
  4. Centrifuge (Eppendorf, model: 5415R )
  5. Centrifuge (Eppendorf, model: 5415D )
  6. Rocker (Fisher scientific, model: 05-450-200 )
  7. 1.7 ml cryptal vial
  8. Bench top mini centrifuge (Thermo Fisher Scientific)
  9. Illumina Eco Real Time PCR system

Procedure

  1. Take 2 x 106 of ATP-depleted or regular IB4 LCL cells and replenish with 10 ml of ATP-depletion or regular culture medium.
  2. Add 274 μl of 36.5% formaldehyde to 10 ml of cells to make the final concentration as 1% and wait 10 min to allow cross link reaction to complete at room temperature.
  3. Add 1 ml of 10x glycine to 10 ml of cells to quench unreacted formaldehyde and swirl gently and incubate at room temperature for 5 min.
  4. Pellet cells at 2,000 rpm (400 x g; Eppendorf 5415D) for 5 min then aspirate supernatant and wash cells twice with 1 ml ice-cold 1x PBS. Finally, add 1 ml ice-cold ChIP lysis buffer and sit on ice for 30 min.
  5. Break down cells on ice by using a douncer homogenizer with 10 strokes and transfer whole cell lysate to a 1.7 ml cryptal vial. Collect nuclei by spin at 2,300 x g for 5 min. After removal of supernatant, resuspend the isolated nuclei with 260 μl digestion buffer (prewarm at 37 °C for 5 min prior to use).
  6. Add 12.5 μl of enzyme solution and allow enzyme digestion at room temperature for 15 min.
  7. Add 20 μl ice-cold EDTA to stop the reaction and pellet the digested chromatins by spining at 13,200 rpm (16,100 x g; Eppendorf 5415R), 4 °C for 10 min. The digested chromatins will be retained in supernatant (around 300 μl: can be applied for up to 5 IP) and cell debris will be left in pellet. Please keep 50 μl of supernatant as input of chromatin DNA.
  8. Aliquot 50 μl of supernatant into 3 new tubes [3 IP: Ab1, Ab2/H3-Ac (positive control), and IgG], and refill each sample with 950 μl ChIP dilution buffer to make a 1 ml volume. Add 20 μl Protein A/G agarose to preclean the non-specifically bound chromatins by sitting on a rocker for 1 h at 4 °C.
  9. After removal of the preclean protein A/G agarose, add 3 μg of the desire Ab1 (in this case PE2), Ab2 for H3-Aceylated (positive control), or IgG (negative control) to the digested chromatins and immunoprecipitation will be carried out by sitting the ChIP samples on a rocker for 4 h at 4 °C.
    Note: Up to 5 IP is allowed.
  10. Add 30 μl Protein A/G agarose to each ChIP sample and allow additional 2 h of reaction at 4 °C. Collect protein A/G agarose by a brief centrifugation using a bench top mini centrifuge.
  11. Wash protein A/G agarose with 1 ml of ice-cold low salt immune complex wash buffer once, 1 ml of ice-cold high salt immune complex wash buffer once, 1 ml of ice-cold LiCl immune complex wash buffer once, and 1 ml of ice-cold TE buffer twice.
  12. Apply 200 μl elution buffer (freshly prepared) for each collected protein A/G agarose sample in order to elute protein by vortexting and incubating at room temperature for 15 min. 200 μl eluent will be obtained for each IP reaction after a brief spin.
  13. Add 8 μl 5 M NaCl to each eluent and incubate at 65 °C from 4 h to overnight to reverse the crosslinking. At this step, the samples can be stored at -20 °C or continue to proceed to the next step.
  14. Add 4 μl 0.5 M EDTA, 8 μl 1 M Tris-HCl, and 1 μl Proteinase K (10 μg/μl) to each sample and incubate at 45 °C for 2 h.
  15. Isolate DNA by using spin columns.
  16. Amounts of DNA will be quantified by performing real time PCR analysis. 5% input DNA will be used as the internal control.

Recipes

  1. ChIP lysis buffer
    20 mM Tris-HCl (pH 8.0)
    85 mM KCl
    0.5% NP40
    Note: Need to add protease inhibitors prior to use.
  2. 10x Glycine
    1.25 M glycine
  3. ChIP dilution buffer
    0.01% SDS
    1.1% Triton X-100
    1.2 mM EDTA
    1.67 mM Tris-HCl (pH 8.1)
    167 mM NaCl
  4. Low Salt Immune Complex Wash Buffer
    0.1% SDS
    1% Triton X-100
    2 mM EDTA
    20 mM Tris-HCl (pH 8.1)
    150 mM NaCl
  5. High Salt Immune Complex Wash Buffer
    0.1% SDS
    1% Triton X-100
    2 mM EDTA
    20 mM Tris-HCl (pH 8.1)
    500 mM NaCl
  6. LiCl Immune Complex Wash Buffer
    0.25 M LiCl
    1% IGEPAL-CA630
    1% deoxycholic acid (sodium salt)
    1 mM EDTA
    10 mM Tris-HCl (pH 8.1)
  7. Elution buffer
    1% SDS
    100 mM NaHCO3
  8. ATP depletion medium
    Glucose (-) RPMI 1640 was supplemented with:
    10% Fetal
    Calf Serum
    100 U/ml Penicillin/100 μg/ml streptomycin
    2 mM Glutamine
    2 mM deoxyglucose
    300 nM antimycin A
  9. Culture medium for IB4 LCL
    RPMI 1640 was supplemented with:
    10% Fetal Calf Serum
    100 U/ml Penicillin/100 μg/ml streptomycin
    2 mM Glutamine
  10. Enzyme solution
    0.125 μl Enzyme stock plus 12.375 μl Glycerol
  11. Protease inhibitors
    1 μg/ml Pepstatin A
    1 μg/ml Aprotinin
    1 μg/ml Leupeptin
    0.5 mM Phenylmethanesulfonyl fluoride
    *Lysis buffer is supplemented with the indicated protease inhibitors

Acknowledgments

This protocol is adapted from Liu et al. (2012).

References

  1. Chen, Y. L., Tsai, H. L. and Peng, C. W. (2012). EGCG debilitates the persistence of EBV latency by reducing the DNA binding potency of nuclear antigen 1. Biochem Biophys Res Commun 417(3): 1093-1099. 
  2. Liu, C. D., Chen, Y. L., Min, Y. L., Zhao, B., Cheng, C. P., Kang, M. S., Chiu, S. J., Kieff, E. and Peng, C. W. (2012). The nuclear chaperone nucleophosmin escorts an Epstein-Barr Virus nuclear antigen to establish transcriptional cascades for latent infection in human B cells. PLoS Pathog 8(12): e1003084. 

简介

EB病毒(EBV)核抗原2(EBNA2)通过模拟激活的Notch受体(Notch-IC)诱导病毒感染的B细胞中病毒和细胞基因的表达,所述Notch受体通过结合重组结合的抑制结构域介导转录激活无毛蛋白抑制剂(RBP-Jκ)。通常,进行染色质免疫沉淀(ChIP)测定,电泳迁移率变动测定(EMSA),链霉亲和素 - 琼脂糖介导的DNA下拉测定以及基于细胞的转录报道基因测定,以验证查询蛋白是否参与EBNA2依赖性转录。核伴侣核蛋白(NPM1)的ATP结合状态已涉及多效生物过程。开发ATP-琼脂糖介导的下拉方案以监测由ATP结合的NPM1诱导的引发前复合物的形成。根据EBNA2和Notch-IC已经显示出在B细胞系中靶基因的活化方面是部分可互换的,可以想象EBNA2是活化的Notch IC的生物学等价物。

第I部分: ATP耗尽

材料和试剂

  1. 葡萄糖( - )RPMI 1640(Life Technologies,目录号:11879020)
  2. 10%胎牛血清(Life Technologies,Gibco )
  3. 青霉素/链霉素(Life Technologies,目录号:15140-122)
  4. 谷氨酰胺(Life Technologies,目录号:25030-081)
  5. 脱氧葡萄糖(Sigma-Aldrich,目录号:D6134)
  6. 抗霉素A(Sigma-Aldrich,目录号:A8674)
  7. RPMI 1640(Life Technologies,目录号:31800089)
  8. ATP消耗介质(参见配方)
  9. IB4 LCL的培养基(参见配方)

设备

  1. CO 2培养箱(Thermo Fisher Scientific Forma CO 2培养箱)
  2. 离心机(Eppendorf,型号:5810R)

程序

  1. 沉淀5×10 6个淋巴母细胞,并补充5ml的ATP消耗培养基。
  2. 在CO 2孵育器(37℃)孵育细胞2小时
  3. 通过以1,050rpm(220×g; Eppendorf 5810R)旋转5分钟收集细胞。
  4. 用1x PBS洗涤细胞两次,并保存进一步的实验目的

食谱

  1. ATP耗尽培养基
    葡萄糖( - )RPMI 1640补充:
    10%胎牛血清
    100U/ml青霉素/100μg/ml链霉素 2mM谷氨酰胺 2mM脱氧葡萄糖 300nM抗霉素A
  2. IB4 LCL培养基
    RPMI 1640补充:
    10%胎牛血清
    100U/ml青霉素/100μg/ml链霉素 2mM谷氨酰胺

参考文献

  1. Liu,   C.D.,Chen,Y.L.,Min,Y.L.,Zhao,B.,Cheng,C.P.,Kang, Chiu,S. J.,Kieff,E。和Peng,C.W。(2012)。 核伴侣 核糖体护送爱泼斯坦 - 巴尔病毒核抗原建立   用于人B细胞中潜在感染的转录级联 Pathog 8(12):e1003084。


第II部分: 链霉亲和素 - 琼脂糖介导的DNA下拉测定

材料和试剂

  1. DNA模板(在这种情况下:LMP1启动子报告质粒)
    注意:转录LMP1启动子对EBV潜伏感染至关重要。
  2. 生物素标记的引物(Vita Genomics)
  3. IB4淋巴母细胞(LCL)
  4. DNA洗脱柱(Geneaid Biotech)
  5. 链霉亲和素结合的琼脂糖(Life Technologies,目录号:SA100-04)
  6. 2×SDS样品缓冲液
  7. NP-40(Sigma-Aldrich,目录号:I8896)
  8. 胃酶抑素A(Sigma-Aldrich,目录号:P5318)
  9. 抑肽酶(Sigma-Aldrich,目录号:A1153)
  10. 亮肽素(Sigma-Aldrich,目录号:L2884)
  11. 苯基甲磺酰氟(Sigma-Aldrich,目录号:P7626)
  12. 裂解缓冲液(见配方)
  13. 蛋白酶抑制剂(参见配方)

设备

  1. PCR产物洗脱柱(Geneaid Biotech)
  2. 离心机(Eppendorf,型号:5810R)
  3. 离心机(Eppendorf,型号:5415R)
  4. 摇臂(管旋转器)
  5. PCR机(MJ Research,型号:PTC 200 Thermal Cycler)
  6. 凝胶电泳设备(Embi Tec,型号:RunOne TM 电泳系统)
  7. Western印迹设备(Bio-Rad,型号:Mini-protean 3)

程序

  1. 通过PCR扩增100μl反应体积中的生物素或非生物素标记的查询启动子的DNA片段
  2. 洗脱   PCR产物通过具有100μl洗脱的洗脱柱 缓冲液,取5μlDNA(5%输入DNA)进行凝胶 电泳
  3. 取1×10 7个淋巴母细胞 用1ml裂解缓冲液裂解,将反应管放在摇床上 在4℃孵育1小时。 通过在13,200rpm下旋转收集上清液 (16,100×g ; Eppendorf 5415R),4℃
  4. 添加所需 生物素标记或非生物素标记的DNA上清并放置管 到摇床上,并在4℃下孵育另一小时
  5. 加 向每个管中加入40μl链霉亲和素结合的琼脂糖,并放入管中 再次在摇杆上,并等待30分钟以允许亲和结合   的链霉亲和素与生物素完成
  6. 通过在1050rpm(220×g /孔; Eppendorf 5810R)中,4℃下旋转5分钟,收获生物素标记的DNA。
  7. 洗   收集链霉亲和素结合的琼脂糖珠与500μl裂解 缓冲液5次,并向每个样品加入40μl2×SDS样品缓冲液 使用它们进行western印迹分析

食谱

  1. 裂解缓冲液(1 L)
    1%NP-40
    2mM EDTA 10%甘油
    50mM Tris-HCl
    150mM NaCl
  2. 蛋白酶抑制剂
    1μg/ml胃酶抑素A
    1μg/ml抑肽酶
    1μg/ml亮肽素 0.5mM苯基甲磺酰氟 *用指定的蛋白酶抑制剂补充裂解缓冲液

参考文献

  1. Liu,   C.D.,Chen,Y.L.,Min,Y.L.,Zhao,B.,Cheng,C.P.,Kang, Chiu,S. J.,Kieff,E。和Peng,C.W。(2012)。 核伴侣 核糖体护送爱泼斯坦 - 巴尔病毒核抗原建立   用于人B细胞中潜在感染的转录级联 Pathog 8(12):e1003084。


第三部分:    ATP-agarose Mediated Pull-Down Analyses

材料和试剂

  1. IB4 LCL细胞
  2. 腺苷5'-三磷酸 - 琼脂糖(Sigma-Aldrich,目录号:A2767-1ml)
  3. 2×SDS样品缓冲液
  4. 葡萄糖( - )RPMI 1640(Life Technologies,目录号:11879020)
  5. 胎牛血清(Life Technologies,Gibco ®
  6. 青霉素/链霉素(Life Technologies,目录号:15140-122)
  7. 脱氧葡萄糖(Sigma-Aldrich,目录号:D6134)
  8. 抗霉素A(Sigma-Aldrich,目录号:A8674)
  9. RPMI 1640(Life Technologies,目录号:31800089)
  10. 谷氨酰胺(Life Technologies,目录号:25030-081)
  11. 胃酶抑素A(Sigma-Aldrich,目录号:P5318)
  12. 抑肽酶(Sigma-Aldrich,目录号:A1153)
  13. 亮肽素(Sigma-Aldrich,目录号:L2884)
  14. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
  15. ATP耗尽培养基(参见配方)
  16. 裂解缓冲液(见配方)
  17. 蛋白酶抑制剂混合物(见配方)
  18. IB4 LCL的培养基(参见配方)

设备

  1. CO <2>孵化器
  2. 离心机(Eppendorf,型号:5415D)
  3. 离心机(Eppendorf,型号:5810R)
  4. 离心机(Eppendorf,型号:5415R)
  5. 摇杆
  6. Western印迹设备( Bio-Rad Laboratories

程序

  1. 搜集   1×10 7个IBL LCL细胞,通过以1,050rpm(220×g)旋转; Eppendorf 5810R),并补加5ml ATP耗尽培养基和 在CO 2培养箱(37℃)中孵育2小时
  2. 收获细胞,并用1x PBS洗涤两次
  3. 溶解   细胞在0.5ml裂解缓冲液中,并将反应管置于摇动器上 并在4℃下孵育1小时。 收集上清液(细胞蛋白)   在13,200rpm(16,100×g; Eppendorf 5415R)上,4℃下10分钟。
  4. 加入50微升ATP-琼脂糖,通过坐在摇臂下拉细胞蛋白,并在4°C孵育4小时。
  5. 搜集   ATP-琼脂糖珠通过在5,000rpm(2,300×g; Eppendorf 5415R) 1分钟,用裂解缓冲液洗涤珠子5次
  6. 向每个收集的下拉样品中加入40μl2x SDS样品缓冲液
  7. 通过蛋白质印迹分析鉴定ATP结合的蛋白质

食谱

  1. 裂解缓冲液(1 L)
    1%NP-40
    2mM EDTA 10%甘油
    50mM Tris-HCl
    150mM NaCl
  2. 蛋白酶抑制剂
    1μg/ml胃酶抑素A
    1μg/ml抑肽酶
    1μg/ml亮肽素 0.5mM苯基甲磺酰氟 *用指定的蛋白酶抑制剂补充裂解缓冲液
  3. ATP消耗培养基
    葡萄糖( - )RPMI 1640补充:
    10%胎牛血清
    100U/ml青霉素/100μg/ml链霉素 2mM谷氨酰胺 2mM脱氧葡萄糖 300nM抗霉素A
  4. IB4 LCL培养基
    RPMI 1640补充:
    10%胎牛血清
    100U/ml青霉素/100μg/ml链霉素 2mM谷氨酰胺

参考文献

  1. Liu,   C.D.,Chen,Y.L.,Min,Y.L.,Zhao,B.,Cheng,C.P.,Kang, Chiu,S. J.,Kieff,E。和Peng,C.W。(2012)。 核伴侣 核糖体护送爱泼斯坦 - 巴尔病毒核抗原建立 用于人B细胞中潜在感染的转录级联 Pathog 8(12):e1003084。


第IV部分:      < Chromatin免疫沉淀(ChIP)

材料和试剂

  1. ATP缺乏的成淋巴细胞样细胞
  2. IB4 LCL细胞(淋巴母细胞样细胞系)
  3. 36.5%甲醛(Sigma-Aldrich,目录号:F8775)
  4. 甘油(Sigma-Aldrich,目录号:F8775)
  5. EDTA(Sigma-Aldrich,目录号:EDS)
  6. 染色质剪切试剂盒(活性基序)
  7. DNA核酸酶(活性基序)
  8. 酶消化缓冲液(Active Motif)
  9. 蛋白A/G琼脂糖(EMD Millipore,目录号:IP10-10ML)
  10. 一抗(Ab1,Ab2和H3-Ac)
    在这种情况下:
    Ab1:EBV核抗原2特异性抗体(例如 Abcam,目录号:ab49498)
    Ab2:核磷蛋白抗体(5E3)(Santa Cruz,目录号:sc-53926)
    Ab3:H3-乙酰化抗体(H3-Ac)(EMD Millipore,目录号:06599)
  11. IgG(Dako,目录号:A0424)
  12. TE缓冲区
  13. 1M Tris-HCl
  14. 蛋白酶K(Life Technologies,目录号:25530049)
  15. 葡萄糖( - )RPMI 1640(Life Technologies,目录号:11879020)
  16. RPMI 1640(Life Technologies,目录号:31800089)
  17. 胎儿小牛血清( Life Technologies,Gibco ®
  18. 青霉素/链霉素(Life Technologies,目录号:15140-122)
  19. 谷氨酰胺(Life Technologies,目录号:25030-081)
  20. 脱氧葡萄糖(Sigma-Aldrich,目录号:D6134)
  21. 抗霉素A(Sigma-Aldrich,目录号:A8674)
  22. ChIP裂解缓冲液(参见配方)
  23. 10x甘氨酸(参见配方)
  24. ChIP稀释缓冲液(参见配方)
  25. 低盐免疫复合物洗涤缓冲液(见配方)
  26. 高盐免疫复合物洗涤缓冲液(参见配方)
  27. LiCl免疫复合物洗涤缓冲液(参见配方)
  28. 洗脱缓冲液(见配方)
  29. ATP消耗介质(参见配方)
  30. IB4 LCL的培养基(参见配方)
  31. 酶溶液(见配方)
  32. 蛋白酶抑制剂(参见配方)

设备

  1. DNA旋转柱(Geneaid Biotech)
  2. Douncer设备(组织研磨机)(Thomas science)
  3. 离心机(Eppendorf,型号:5810R)
  4. 离心机(Eppendorf,型号:5415R)
  5. 离心机(Eppendorf,型号:5415D)
  6. Rocker(Fisher scientific,型号:05-450-200)
  7. 1.7 ml cryptal小瓶
  8. 台式迷你离心机(Thermo Fisher Scientific)
  9. Illumina Eco实时PCR系统

程序

  1. 取2×10 6个缺乏ATP或正常的IB4 LCL细胞,并补充10ml的ATP耗竭或常规培养基。
  2. 加   274微升36.5%甲醛至10毫升细胞做最后 浓度为1%,并等待10分钟以使交联反应 在室温下完成
  3. 加入1ml的10x甘氨酸至10   ml细胞淬灭未反应的甲醛,轻轻摇动 在室温下孵育5分钟
  4. 颗粒细胞 (400×g /孔; Eppendorf 5415D)5分钟,然后吸出上清液   并用1ml冰冷的1×PBS洗涤细胞两次。 最后,加入1ml 冰冷的ChIP裂解缓冲液,并在冰上放置30分钟
  5. 打破   通过使用douncer匀浆器用10次冲击在冰上下降细胞 将全细胞裂解物转移至1.7ml隐窝小瓶中。 收集核 在2,300×g下旋转5分钟。 去除上清液后,重悬   用260μl消化缓冲液(在37℃预热5分钟)分离核 min)。
  6. 加入12.5μl酶溶液,并在室温下酶消化15分钟
  7. 加   20μl冰冷的EDTA停止反应,沉淀消化 通过在13,200rpm(16,100×g /孔; Eppendorf 5415R),4℃ 10分钟。 消化的chromatins将保留在上清液中 (约300μl:可应用于多达5个IP)和细胞碎片 留在沉淀。 请保留50微升的上清液作为染色质的输入 脱氧核糖核酸。
  8. 等分50μl上清液到3个新管[3 IP: Ab1,Ab2/H3-Ac(阳性对照)和IgG],并重新填充每个样品   950μlChIP稀释缓冲液,使1 ml体积。 加入20μl蛋白 A/G琼脂糖通过坐下预清洁非特异性结合的染色质   在4℃下摇动1小时。
  9. 去除预清洁剂后   蛋白A/G琼脂糖,加入3μg期望的Ab1(在这种情况下为PE2),Ab2   对于H3-Aceylated(阳性对照)或IgG(阴性对照) 消化的chromatins和免疫沉淀将进行 将芯片样品在摇床上在4℃下放置4小时 注意:最多允许5个IP。
  10. 加 30μl蛋白A/G琼脂糖到每个ChIP样品,并允许另外2小时 的反应在4℃。 收集蛋白A/G琼脂糖短暂 使用台式迷你离心机离心
  11. 洗 蛋白A/G琼脂糖与1ml冰冷的低盐免疫复合物洗涤 缓冲液一次,1ml冰冷的高盐免疫复合物洗涤缓冲液一次,   1ml冰冷的LiCl免疫复合物洗涤缓冲液一次,和1ml 冰冷的TE缓冲液两次。
  12. 应用200μl洗脱缓冲液 (新鲜制备)每种收集的蛋白A/G琼脂糖样品 以便通过在室温下涡旋和温育来洗脱蛋白质 15分钟。 a后,每次IP反应得到200μl洗脱液 短暂旋转。
  13. 向每个洗脱液中加入8μl5 M NaCl,并孵育 在65℃下4小时至过夜,以逆转交联。 在这 步骤,样品可以储存在-20°C或继续进行 下一步。
  14. 向每个样品中加入4μl0.5 M EDTA,8μl1M Tris-HCl和1μl蛋白酶K(10μg/μl),并在45°C孵育2小时。
  15. 使用旋转柱分离DNA。
  16. 通过进行实时PCR分析来定量DNA的量。 5%输入DNA将用作内部对照

食谱

  1. ChIP裂解缓冲液
    20mM Tris-HCl(pH8.0) 85 mM KCl
    0.5%NP40
    注意:使用前需要加入蛋白酶抑制剂。
  2. 10x甘氨酸
    1.25 M甘氨酸
  3. ChIP稀释缓冲液
    0.01%SDS
    1.1%Triton X-100 1.2mM EDTA
    1.67mM Tris-HCl(pH8.1) 167 mM NaCl
  4. 低盐免疫复合物洗涤缓冲液
    0.1%SDS
    1%Triton X-100 2mM EDTA 20mM Tris-HCl(pH8.1) 150mM NaCl
  5. 高盐免疫复合物洗涤缓冲液
    0.1%SDS
    1%Triton X-100 2mM EDTA 20mM Tris-HCl(pH8.1) 500 mM NaCl
  6. LiCl免疫复合物洗涤缓冲液
    0.25 M LiCl
    1%IGEPAL-CA630
    1%脱氧胆酸(钠盐)
    1mM EDTA
    10mM Tris-HCl(pH8.1)
  7. 洗脱缓冲液
    1%SDS
    100mM NaHCO 3/v/v
  8. ATP耗尽培养基
    葡萄糖( - )RPMI 1640补充:
    10%胎儿
    小牛血清
    100U/ml青霉素/100μg/ml链霉素 2mM谷氨酰胺 2mM脱氧葡萄糖 300nM抗霉素A
  9. IB4 LCL培养基
    RPMI 1640补充:
    10%胎牛血清
    100U/ml青霉素/100μg/ml链霉素 2mM谷氨酰胺
  10. 酶溶液
    0.125μl酶储备液加上12.375μl甘油
  11. 蛋白酶抑制剂
    1μg/ml胃酶抑素A
    1μg/ml抑肽酶
    1μg/ml亮肽素 0.5mM苯基甲磺酰氟 *用指定的蛋白酶抑制剂补充裂解缓冲液

致谢

该协议改编自Liu等人(2012)。

参考文献

  1. Chen,Y.L.,Tsai,H.L.和Peng,C.W。(2012)。 EGCG通过降低核抗原1的DNA结合效能使衰弱EBV潜伏期的持续性。 Biochem Bi ophys Res Commun 417(3):1093-1099。 
  2. Liu,   C.D.,Chen,Y.L.,Min,Y.L.,Zhao,B.,Cheng,C.P.,Kang, Chiu,S. J.,Kieff,E。和Peng,C.W。(2012)。 核伴侣 核糖体护送爱泼斯坦 - 巴尔病毒核抗原建立   用于人B细胞中潜在感染的转录级联 Pathog 8(12):e1003084。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Liu, C., Chen, Y., Min, Y., Zhao, B., Cheng, C., Kang, M., Chiu, S., Kieff, E. and Peng, C. (2013). Chromatin Immunoprecipitation (ChIP), Streptavidin and ATP-agarose Mediated Pull-down Analyses. Bio-protocol 3(18): e901. DOI: 10.21769/BioProtoc.901.
  2. Liu, C. D., Chen, Y. L., Min, Y. L., Zhao, B., Cheng, C. P., Kang, M. S., Chiu, S. J., Kieff, E. and Peng, C. W. (2012). The nuclear chaperone nucleophosmin escorts an Epstein-Barr Virus nuclear antigen to establish transcriptional cascades for latent infection in human B cells. PLoS Pathog 8(12): e1003084.
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