Like use other ChIP kits, before handle ChIP assay please choose a good antibody suitable for precipitation the crosslinked protein / DNA complexes.

Transcription

1 ChIP Assay Kit Cat:RK20100 Like use other ChIP kits, before handle ChIP assay please choose a good antibody suitable for precipitation the crosslinked protein / DNA complexes. Manufactured by Global Headquarters 86 Cummings Park Woburn, MA Tel: China Branch 388# Gaoxin Road (No.2) Tel: East Lake Development Zone market@abclonal.com Wuhan P. R. China http: 1

2 Contents Introduction... 3 Principle of the Assay... 4 Materials Provided... 5 Flow Chart of Process... 6 References... 7 Precautions For Use... 8 Additional materials required... 9 Assay Procedure Section A. Chromatin Crosslink with Cultured Cells Section B. Chromatin Crosslink with Tissue Samples Section C. Chromatin Fragmentation Section D. Chromatin Immunoprecipitation Section E. Reverse Corsslink and DNA Purification Kit Performance and Benefits Appendix Section A. ChIP-qPCR Primer Design Section B. Troubleshooting Guide

3 Introduction Genome wide mapping of protein/dna interactions and epigenetic marks is essential for a full understanding of transcriptional regulation. A precise map of binding sites for histone modifications, transcription factors, core transcriptional machinery and other DNA binding proteins is crucial for deciphering the gene regulatory networks that underlie various biological processes. Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation technique, which is a common technique for studying epigenetics, used to investigate the interaction between protein and DNA in the nucleus of cells. Chromatin immunoprecipitation (ChIP) technology has provided valuable information regarding protein/dna interactions, which has increased our understanding of the functional organization of the genome. In ChIP assay, DNA binding proteins are crosslinked to DNA by formaldehyde then chromatin is fragmented into bp range. An antibody specific to the protein of interest is used to co-immunoprecipitate protein/dna complex. Finally, the protein/dna crosslink are reversed and DNA is purified for downstream analysis. 3

4 Principle of the Assay Chromatin is composed of DNA and histones, nervertheless many transcription factors or co-factors also bind to chromatin and regulate gene expression. Chromatin immunoprecipitation (ChIP) is a powerful tool for studying protein/dna interactions. This ChIP assay kit contains all reagents for carrying out a successful chromatin immunoprecipitation in mammalian tissue samples or cultured cells. When performing ChIP assay, cells are fixed by formaldehyde, a small chemical molecule servers to crosslink protein and DNA. Cells are then lysed to release nucleus, and chromatin is fragmented using micrococcal nuclease digestion or sonication. Fragmented chromatin is then subjected to ChIP using specific ChIP grade antibodies against histone modifications or transcription factors. DNA and protein are co-precipitate as a complex, protein is then digested using proteinase K and DNA is purified. Sequence information of ChIP DNA could be detected by a mount of methods, such as PCR, qpcr, DNA sequencing, microarray, southern blot and high throughput sequencing. This ChIP assay kit contains sufficient reagents to prepare 22 chromatin ChIP reactions. 4

5 Materials Provided Reagents Quantity Storage Lysis Buffer 30 ml Room temperature Dilution Buffer 30 ml Room temperature Wash Buffer I 30 ml Room temperature Wash Buffer II 30 ml Room temperature Wash Buffer III 30 ml Room temperature TE Buffer 48 ml Room temperature Elution Buffer 5 ml Room temperature Protein A/G Plus Magpoly Beads 1.5 ml 4 degree 10X Glycine buffer 15 ml 4 degree 1000X Protease Inhibitor Cocktail 25 ul -20 degree Proteinase K 350 ul -20 degree Control Primers 200 ul of 5 um -20 degree Anti-H3 25 µg -20 degree Rabbit IgG 25 µg -20 degree 5

6 Flow Chart of Process Cells are fixed by formaldehyde to crosslink protein and DNA. Cells are then lysed to release nucleus and chromatin is fragmented by sonication. Antibodies directed against the protein or histone modification of interest is conjugated to protein A/G beads for immunoprecipitation of the DNA of interest. Following reverse crosslink and elution from beads, DNA is purified and processed to downstream analysis, such as qpcr, microarray and high throughput sequencing. 6

7 References Park, Peter J. "ChIP seq: advantages and challenges of a maturing technology." Nature Reviews Genetics (2009): Gade, Padmaja, and Dhan V. Kalvakolanu. "Chromatin ChIP assay as a tool for analyzing transcription factor activity." Transcriptional Regulation: Methods and Protocols (2012): Kuo, Min-Hao, and C. David Allis. "In vivo cross-linking and immunoprecipitation for studying dynamic protein: DNA associations in a chromatin environment." Methods 19.3 (1999):

8 Precautions For Use 1. Reagents may be harmful, if ingested, rinse it with an excess amount of tap water. 2. Formaldehyde solution is a highly toxic chemical, safety glasses, gloves and lab coat should be used. The fixation step should be handled in ventilated hood. 3. All components should be stored in right temperature according to the specification refrigerated. 4. Please perform simple centrifugation to collect the liquid before use. 5. The kit should not be used beyond the expiration date. 6. To avoid cross contamination, please use disposable pipette tips. 8

9 Additional materials required 1. 37% formaldehyde solution. 2. Custom ChIP grade antibody directed against the protein of interest. 3. Phosphate buffer saline (PBS) solutions. 4. Bovine Serum Albumins (BSA). 5. DNA purification kit. 6. Magnetic stand. 7. Cell scraper. 8. Appratus to rotate tubes at 4 degree. 9. Microcentrifuge and microcentrifuge tubes. 10. Pipettors and tips (filter tips are recommended). 11. Agarose gel electrophoresis apparatus. 12. Tissue homogenizer apparatus. 9

10 Assay Procedure Section A. Chromatin Crosslink with Cultured Cells Crosslink and chromatin shearing from 10cm dish (approximately 1 x 10 7 cells) involved in the protocol. This shearing protocol assumes that you have already optimized shearing conditions for your specific cell line and treatment. 1. Grow cells in 10cm dish, and harvest cells when grow to 70-80% confluency. 2. Add 275ul 37% formaldehyde to 10ml cell culture medium and mix thoroughly, leave at room temperature, incubate on a shaking platform for 10 minutes. 3. Stop the crosslink reaction by adding 500ul glycine solution (10x glycine solution), incubate at room temperature for 5 minutes on a shaking platform. 4. Wash the plate by discard glycine stop fixation buffer, then adding 1ml prior ice-cold 1 x PBS buffer. Rock the plate gently then pouring off the PBS. 5. Harvest cells by scraper, spin down at 1000 x g for 5 minutes at 4 degree then discard supernatant. 6. Wash cells with 1ml ice-cold 1 x PBS, spin down at 1000 x g for 5 minutes at 4 degree, then discard supernatant. 7. Aspirate PBS, flash freeze and keep the cell pellet frozen at

11 degree until needed. Note: In standards protocol, chromatin crosslink for 10 minute by 1% concentration formaldehyde prior shearing while some protein/dna combinations may work better with shorter fixation time. Section B. Chromatin Crosslink with Tissue Samples 1. For each sample, choose mg fresh or frozen tissue sample. Cut the tissue into small pieces (approximately 1mm cubes) using a razor blade. 2. Transfer tissue pieces into a tube, place the tube on ice and homogenize the contents by tissue homogenizer. 3. Pellet the cells from step 2 by centrifugation for 5 minutes at 1000 x g at 4 degree. 4. Remove the supernatant and discard. Resuspend the pellet in 10ml prior ice-cold PBS. 5. Add 275ul 37% formaldehyde to cells from step 4 and mix thoroughly, leave at room temperature, incubate on a shaking platform for 10 minutes. 6. Stop the crosslink reaction by adding 500ul glycine solution(10x glycine solution), incubate at room temperature for 5 minutes on a shaking platform. 7. Pellet the cells from step 6 by centrifugation for 5 minutes at 1000 x g at 4 degree. 8. Wash cells with 1ml ice-cold 1 x PBS, spin down at 1000 x g for 5 11

12 minutes at 4 degree, then discard supernatant. 9. Aspirate PBS, flash freeze and keep the cell pellet frozen at -80 degree until needed. Note: In standards protocol, chromatin crosslink for 10 minute by 1% concentration formaldehyde prior shearing while some protein/dna combinations may work better with shorter fixation time. Section C. Chromatin Fragmentation 1. Add 200ul cell Lysis Buffer containing 1ul protease inhibitor (1000x Protease Inhibitor Cocktail), Incubate for 30 minutes on a rocker at 4 degree. 2. Add 800ul ChIP Dilution Buffer to final volume 1ml. 3. Chromatin shearing by microtip probe sonicator: a) Amplitude 30-50% b) Process time minutes c) Pulse on time 0.5s d) Pulse off time 0.5s 4. After sonication, centrifuge at x g for 15 minutes at 4 degree. Transfer the supernatant to a new tube. 5. Transfer 50ul supernatant as input, add 50ul Dilution Buffer and 5ul Proteinase K, incubate at 65 degree for 4 hours or overnight to reverse crosslink. 6. Purify reverse corsslinked DNA using DNA purification kit, elute by 50ul Elution Buffer. 12

13 7. Load 5ul input DNA on a 2% agarose gel to check chromatin shearing performance. 8. Fragmented chromatin could be store at -80 degree until needed. Note: For different cell types and stimulation conditions, chromatin shearing programs may be different. Prior ChIP assay, chromatin shearing conditions should be optimized. Section D. Chromatin Immunoprecipitation 1. Wash 60ul Protein A/G Plus Magpoly Beads by 1ml ice-cold 1 x PBS. 2. Place the tube on magnetic particle collector for 2 minutes, then discard supernatant. 3. Add BSA to block Protein A/G Plus Magpoly Beads, incubate at 4 degree for 5 minutes. 4. Place tube on magnetic particle collector for 2 minute, then discard supernatant. 5. Wash beads by adding 1ml TE Buffer and mix gently, then discard supernatant. 6. Add 100ul TE Buffer and 3-5 ug antibody, incubate at 4 degree for 2 hours to allow complete antibody binding to beads. 7. Add 1ml chromatin extracts to the beads, incubate at 4 degree on a rocker for overnight. 13

14 Section E. Reverse Corsslink and DNA Purification 1. Wash beads by adding 1ml ice-cold Wash buffer I, incubate at 4 degree on a rocker for 5 minutes. 2. Place tube on a magnetic particle collector for 2 minute then discard supernatent. 3. Wash beads by adding 1ml ice-cold Wash buffer II, incubate at 4 degree on a rocker for 5 minutes. 4. Place tube on a magnetic particle collector for 2 minute then discard supernatant. 5. Wash beads by adding 1ml ice-cold Wash buffer III, incubate at 4 degree on a rocker for 5 minutes. 6. Place tube on a magnetic particle collector for 2 minute then discard supernatant. 7. Wash beads by adding 1ml ice-cold TE buffer, incubate at 4 degree on a rocker for 5 minutes. 8. Place tube on a magnetic particle collector for 2 minute then discard supernatant. 9. Add 200ul ChIP Elution Buffer and 10ul Proteinase K, incubate at 65 degree for 4 hours or overnight to reverse crosslink. 10. After reverse crosslink, place tube on magnetic particle collector for 2 minutes then transfer supernatant to a new tube. 11. Purify reverse corsslinked DNA, elute by ul Elution Buffer. 12. ChIP DNA could be used for ChIP-qPCR detection or store at -80 degree until used. 14

15 Kit Performance and Benefits Fig1. Chromatin fragmentation efficiency in NIH3T3 and HaCaT cells line. Chromatin sonication procedure was handled as the manufacture s recommendation. Briefly, cells were crosslinked by formaldehyde and then stopped by glycine. After scrapped from plate, cells were lysed by lysis buffer to release nucleus, and chromatin fragmented by sonication. Chromatin reversed crosslink at 65 degree with proteinase K to digest protein. DNA was purified by DNA purification kit and gel electrophoresis was used to test chromatin fragmentation efficiency. 15

16 % Input HCT116 CTCF c-myc 1 c-myc 2 H19/igf2 1 H19/igf2 2 α-satellite IgG H243 Fig2. ChIP assay for CTCF in HCT116 cell line. ChIP was performed according to the manufacture s recommendation. Abclonal CTCF antibody, which was validated as ChIP grade, was used to pull down the specific chromatin of interest. Primer was designed from ENCODE CTCF ChIP-Seq data in UCSC genome browser. 16

17 Appendix Section A. ChIP-qPCR Primer Design Chromatin immunoprecipitation is a tool for decipher protein/dna binding sites. Success of ChIP assay depends on good primers. In general, optional ChIP-qPCR primer range from 20bp to 30bp, and the products should be bp. Too long primers and products should be avoid which will yield amplification efficiency. Nowadays, thers are several projects and databases support ChIP-Seq profilings for most popular histone modifications and DNA binding proteins in human and mouse. We recommend you to use ENCODE data in UCSC genome browser or Roadmap data to choose a proper ChIP primer. man.html 17

18 Section B. Troubleshooting Guide The amount of cell and tissue for ChIP Measuring the amount of cells and tissue for ChIP assay is very import. We recommend using x 10 7 cells or mg tissue for ChIP. Too few cells or tissue will yield low concentration chromatin. Generally, we recommend 7-10ug of chromatin for histone modifications and high abundance transcription factors (TFs). For low abundance of TFs, 10ug chromatin is needed at least. We do not recommend using large amount of chromatin as the high concentration chromatin always yield sonication efficiency and need more amount of antibody for precipitation. Poor yield of sheared chromatin efficiency There are several reasons for poor yield of chromatin shearing efficiency. Formaldehyde fixation time will influence chromatin structure and cause over crosslink. Over fixed cells are often resistant to lysis and chromatin fragmentation. Cell lysis time is also very import. For high efficient chromatin fragmentation, cell nuclei should be released, the more nuclei release the higher the sheared chromatin yield. Sonication volume We recommend the minimal sonication volumn is 400ul and maximal volume is 1000ul. For a large amount of cells or samples, we strongly suggest to divided into small volumn for sonication, and then pool the samples at the end. 18

19 Optimize ChIP antibody A ChIP grade antibody is recommended for this assay. Since the fixation step will influence chromatin structure and block apitope. Validate whether antibody is ChIP grade is very important. High background The step of formaldehyde fixation is essential for ChIP. Over crosslink of DNA and protein always yield high background. Chromatin shearing efficiency is also very important. If the chromatin fragment is too large, there is a high possibility your amplicon from neighbour of the true binding sites. We suggest the best chromatin fragmentation size range from 200bp to 800bp. The antibodies for ChIP assay should be validate as ChIP or IP grade and we do not recommend used excess antibody for immunoprecipitation which will result in more non-specific binding. Pre-block the magnetic beads by BSA or Salmon sperm DNA can also decrease background. Low DNA concentration The amount of chromatin as initiation will influence final DNA concentration. This ChIP assay kit is not suit for small scale ChIP and the amount of chromatin for immunoprecipitation is no less than 5ug. The quality and quantity of antibody are the key for ChIP assay, we recommend using ChIP or IP grade antibodies and 3-5ug antibody per 10ug chromatin. 19