EZ-ChIP™
Chromatin Immunoprecipitation Kit
Catalog # 17-371
Instruction Manual
Sufficient reagents for 22 chromatin immunoprecipitation (ChIP) assays per kit.
Contents
Page
I. INTRODUCTION
哈尔滨化妆培训学校2 II. CHROMATIN IP ASSAY OVERVIEW
4 A. Chromatin Sample Prep and Immunolection 4 B. DNA Purification and Detection 4 III.
EZ ChIP™ KIT COMPONENTS 5 A. Provided Kit Components
5 B. Required Materials Not Provided
6 IV. CHROMATIN IMMUNOPRECIPITATION PROTOCOL
7 A. In Vivo Crosslinking and Lysis 7 B. Sonication to Shear DNA
8 C. Immunoprecipitation (IP) of Crosslinked Protein/DNA 8 D. Elution of Protein/DNA Complexes 10 E. Rever Crosslinks of Protein/DNA Complexes to Free DNA 10 F. DNA Purification using Spin Columns 10 G. PCR of Controls
11 V. APPENDIX A – Optimization of DNA Sonication 14 VI. APPENDIX B – Formaldehyde Preparation
16 VII. CHROMATIN IP OPTIMIZATION AND TROUBLESHOOTING
17
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FOR RESEARCH USE
ONLY.
DO NOT USE IN DIAGNOSTIC PROCEDURES.
DO NOT USE IN HUMANS.
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I. INTRODUCTION
Chromatin Immunoprecipitation (ChIP) is a widely ud method to identify specific proteins associated with a region of the genome, or in rever, to identify regions of the genome associated with specific proteins. The proteins can be isoforms of histones modified at a particular amino acid or other chromatin-associated proteins. When employed with antibodies that recognize histone modifications, ChIP can be ud to “measure” the amount of the modification. An example of this would include measurement of the amount of histone H3 acetylation associated with a specific gene promoter region under various conditions that might alter expression of the gene. Histones are not the only proteins that can be studied using this technique. Much of the recent interest has been in analyzing transcription factor distribution throughout the genome or at specific loci.
When performing ChIP, cells are first fixed with formaldehyde to covalently crosslink proteins to DNA. Then chromatin is harvested from the cells and subjected to an immunolection process, which requires the u of specific antibodies. Any DNA quences cross-linked to the protein of interest will co-precipitate as part of the chromatin complex. After the immunolection of chromatin fragments and purification of associated DNA, the detection of specific DNA quences is performed. If the DNA which will be detected is associated with the protein or histone modification being examined, the relative reprentation of that DNA quence will be incread (or enriched) by the immunoprecipitation process. Generally, standard PCR is performed to identify the DNA quence (the gene or region of the genome) associated with the protein of interest. The relative abundance of a specific DNA quence isolated via the protein-specific immunolection is compared to DNA obtained when using an unrelated antibody control. DNA fragments are run on gels to facilitate quantitation of the PCR products. A much more accurate alternative to standard PCR is real time quantitative PCR (RT-qPCR). Cloning of quences from a ChIP experiment is also possible, to create libraries of fragments that are enriched for tho that interact with a particular protein. The combination of chromatin IP with microarray applications (ChIP on chip) is a novel technique that is becoming more popular, allowing the generation of genome-wide maps of protein-DNA interactions or histone modifications.
The EZ ChIP™ kit contains the buffers and reagents required to perform a successful ChIP from mammalian cells. Importantly, EZ ChIP™ also contains esntial controls (anti-RNA Polymera II, Normal Mou IgG and Control Primers) to ensure that the ur has successfully performed the ChIP assay. RNA Polymera II is responsible for the transcription of protein coding genes, and therefore, is prent at the promoter region of genes that are actively transcribed. The glyceraldehyde-3-phosphate dehydrogena (GAPDH) gene is considered a houkeeping gene and expected to be undergoing transcription in most growing mammalian cells. Upon immunoprecipitation of chromatin with an antibody to RNA Polymera II, the resulting DNA is enriched for the GAPDH gene (as well as all transcribed genes), whereas immunoprecipitation with Normal Mou IgG will not result in GAPDH enrichment. The covalent bonds between the DNA and associated proteins are then vered, and the DNA is purified prior to performing PCR. For DNA purification, the EZ ChIP™ kit incorporates a unique polypropylene spin column manufactured by Mo Bio Laboratories, Inc. Each spin column contains a specially activated silica membrane filter that captures DNA and parates it from contaminating proteins and other cellular debris. After soluble contaminants are spun through the filter, the column is washed and then DNA is eluted in a low-salt buffer. Bind Buffer “A”, Wash Buffer “B”, and Elute Buffer “C” are supplied by Mo Bio Laboratories and are RNa and DNa free. This technology from Mo Bio Laboratories, Inc. provides rapid purifi
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cation of chromatin DNA without the need for phenol chloroform extractions or ethanol precipitation. The purified DNA is subjected to PCR using the Control Primers which are specific to the promoter region of the GAPDH gene.
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Kit Description
Quantity: Two boxes containing the necessary reagents to perform 22 chromatin immunoprecipitation (ChIP) assays. This kit also contains reagents to generate chromatin from five 15-cm plates, each of which provides sufficient chromatin for up to 10 individual precipitations
Storage and Stability: Upon receipt, store components at the temperatures indicated on the labels.
大学英语精读第二册答案Storage temperatures are also indicated on page 5 of this manual. Kit components are stable for 1 year from date of shipment when stored as directed.
U: The EZ ChIP™ kit contains reagents optimized for immunoprecipitation of chromatin from mammalian cells and includes controls to ensure the successful performance of this assay. The included positive control antibody is a mou monoclonal antibody to RNA Polymera II and will detect RNA Polymera II of human, mou, rat and yeast origins. The negative control is Normal Mou IgG, which controls for the non-specific immunolection of chromatin by immunoglobulins. Control Primers are included for detection of a 166 ba pair region of the human GAPDH promoter by PCR. U of the primers for DNA from other species has not been evaluated. Detection of the DNA region, gene or promoter of interest in immunoprecipitated chromatin must be empirically determined by the rearcher. PCR using promoter-specific primers is recommended for detection and analysis of enriched DNA.
The EZ ChIP™ kit has all the necessary buffers and reagents to perform successful chromatin immunoprecipitation assays, however, careful attention must be paid to the details of the instructions. Follow all the instructions carefully, especially with regard to incubation times and temperatures.
Related Products:
Catalog # 17-295 Chromatin Immunoprecipitation Kit Catalog # 17-375 EZ-Zyme Chromatin Preparation Kit Catalog # 17-245 Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit Catalog # 17-229 Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit Catalog # 16-157 Protein A agaro/Salmon Sperm DNA Catalog # 16-201 Protein G agaro/Salmon Sperm DNA Catalog # 17-610 Magna ChIP™ A Chromatin Immunoprecipitation Kit Catalog # 17-611 Magna ChIP™ G Chromatin Immunoprecipitation Kit Catalog # 17-408 EZ-Magna ChIP™ A Chromatin Immunoprecipitation Kit Catalog # 17-409 EZ-Magna ChIP™ G Chromatin Immunoprecipitation Kit
For a complete list of Millipore’s ChIP qualified antibodies, go to and arch “ChIP”.
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II.
CHROMATIN IP ASSAY OVERVIEW
A. Chromatin Sample Prep and Immunolection
Grow cells and treat with formaldehyde. This treatment crosslinks the proteins to the DNA, ensuring co-precipitation of the DNA with the protein of interest.
Lysis and sonication of the cells. Cells are broken open and sonication is performed to shear the chromatin to a manageable size. Generally, 200-1000bp of DNA is small enough to achieve a high degree of resolution during the detection step. It is critical that average fragment size is confirmed empirically by gel electrophoresis.
Immunolection. This step is very similar to a standard immunoprecipitation, which us a primary antibody of choice followed by Protein G-conjugated agaro beads as the condary reagent. This enriches for the protein of interest and the DNA that is specifically complexed with it.
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B. DNA Purification and Detection
Purification of the DNA. Protein-DNA crosslinks are reverd during incubation at 65°C and DNA is purified to remove the chromatin proteins and to prepare the DNA for the detection step.
Detection. This is the most variable step of the procedure becau of the number of detection methods that can be employed and the variability of PCR primer lection. The most meaningful results will be obtained with quantitative PCR for this step. Real Time Quantitative PCR (RT-qPCR) is ideal, but this method requires a specialized PCR machine that may not be available. For standard PCR, primer lection is critical and must be designed with clo adherence to the following guidelines:
Primer Length: 24 nt Optimum Tm: 60°C Optimum GC: 50% Amplicon size: 100-700 ba pairs
After standard PCR, the fragments are run on agaro or polyacrylamide gels, and the gels are stained
and imaged as appropriate.
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III.
EZ ChIP™ KIT COMPONENTS
A. Provided Kit Components (Note Storage Temperatures)
Store at 4°C:
ChIP Blocked Protein G Agaro, Catalog # 16-201D. One vial containing 1.5 mL packed beads with 1.5 mg BSA and approximately 4.5mg recombinant Protein G. Provided as a 50% gel slurry for
a final volume of 3 mL per vial. Suspended in TE buffer, pH 8.0, containing 0.05% sodium azide. Liquid suspension. ChIP Dilution Buffer, Catalog # 20-153. One vial containing 24 mL .
Low Salt Immune Complex Wash Buffer, Catalog # 20-154. One vial containing 24 mL . High Salt Immune Complex Wash Buffer, Catalog # 20-155. One vial containing 24 mL . LiCl Immune Complex Wash Buffer, Catalog # 20-156. One vial containing 24 mL .
TE Buffer, Catalog # 20-157. Two vials, each containing 24 mL.
0.5 M EDTA, Catalog # 20-158. One vial containing 250 µL .
5 M NaCl, Catalog # 20-159. One vial containing 500 µL .
腐化堕落SDS Lysis Buffer, Catalog # 20-163. One vial containing 10 mL .
1 M Tris-HCl, pH 6.5, Catalog # 20-160. One vial containing 500 µL.
10X Glycine, Catalog # 20-282. One vial containing 11 mL .
10X PBS, Catalog # 20-281. One vial containing 24 mL .英语手抄报图片大全
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Store at -20°C:
Protea Inhibitor Cocktail II, Catalog # 20-283. Two vials, each containing 110 µL of 200X Protea Inhibitor Cocktail II in DMSO. RNa A, Catalog # 20-297. One vial containing 600 µg of RNa A in 60 µL sterile water.
Proteina K, Catalog # 20-298. One vial containing 600 µg of Proteina K in 60 µL. 1M NaHCO 3, Catalog # 20-296. One vial containing 600 µL .
Control Primers, Catalog # 22-004. One vial containing 75 µL of 5 µM of each control primer specific for human GAPDH.
FOR: 5'-TACTAGCGGTTTTACGGGCG-3’ REV: 5'-TCGAACAGGAGGAGCAGAGAGCGA-3'
Anti-RNA Polymera II, Catalog # 05-623B. One vial containing 25 µg of Anti-RNA Polymera II, clone CTD4H8.
Normal Mou IgG, Catalog # 12-371B. One vial containing 25 µg of normal mou IgG.
Store at Room Temperature:
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20% SDS, Catalog # 20-280. One vial containing 242 µL of 20% SDS.
Spin Filters, Catalog # 20-290. One bag containing 22 Spin Filters with Collection Tubes. Collection Tubes , Catalog # 20-291. One bag containing 22 Collection Tubes.
Bind Reagent A, Catalog # 20-292. One vial containing 25 mL of Bind Reagent A.
Wash Reagent B, Catalog # 20-293. One vial containing 12.5 mL of Wash Reagent B. Elution Reagent C, Catalog # 20-294. One vial containing 1.5 mL of Elution Reagent C.
