Chromatin Immunoprecipitation Assay: A Technique to Study Protein-DNA Interactions in Cells
Published: April 30, 2023
Abstract
Source: Fuchs, A. R.,et al. A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia. J. Vis. Exp. (2018).
This video describes protocol for chromatin immunoprecipitation (ChIP) assay, which is a classic technique to demonstrate protein-DNA interactions.This technique is used to identify target genes in human chronic lymphocytic leukemia cells.
Protocol
1. Chromatin Immunoprecipitation NOTE: The chromatin immunoprecipitation was performed with a commercially available ChIP kit according to the manufacturer's instructions with modifications. Calculate the number of immunoprecipitations (70 µL (Jurkat cells) or 15 µL (patient samples) of sheared chromatin plus one antibody) and prepare 20 µL of protein A coated beads per precipitation in a 1.5 mL tube. Wash the be…
Divulgaciones
The authors have nothing to disclose.
Materials
| Anti-NFAT2 antibody | Alexis | 1008505 | Clone 7A6 |
| Anti-NFAT2 antibody | Cell Signaling | 8032S | Clone D15F1 |
| Anti-NFAT2 antibody ChIP Grade | Abcam | ab2796 | Clone 7A6 |
| Magnetic rack, DynaMag-15 Magnet | Thermo Scientific | 12301D | Can be substituted with similar instruments |
| iDeal ChIp-seq kit for Histones | Diagenode | C01010059 | |
| Small Centrifuge | Thermo Scientific | Heraeus Fresco 17 | Can be substituted with similar instruments |
| Shaker Duomax 1030 | Heidolph Instruments | 543-32205-00 | Can be substituted with similar instruments |
| Halt Protease and Phosphatase Inhibitor Cocktail (100X) | Thermo Scientific | 78440 | |
| IRDye 680LT Donkey anti-Rabbit IgG (H + L), 0.5 mg | LI-COR Biosciences | 926-68023 | |
| IRDye 800CW Goat anti-Mouse IgG (H + L), 0.1 mg | LI-COR Biosciences | 925-32210 | |
| DNA LoBind Tube 1.5 mL | eppendorf | 22431021 | |
| FBS superior | Merck | S0615 | |
| 37 % Formaldehyde p.a., ACS | Roth | 4979.1 | |
| 1 X PBS | Sigma Aldrich | D8537 | |
| M220 Focused-ultrasonicator | Covaris | 500295 | |
| 20X Bolt MES SDS Running Buffer | Thermo Scientific | B0002 | |
| Tris Buffered Saline (TBS-10X) | Cell Signaling | #12498 | |
| iBlot 2 Gel Transfer Device | Thermo Scientific | IB21001 | |
| iBlot 2 Transfer Stacks, nitrocellulose, regular size | Thermo Scientific | B23001 | |
| big Centrifuge | Eppendorf | 5804R | Can be substituted with similar instruments |
Citar este artículo
Chromatin Immunoprecipitation Assay: A Technique to Study Protein-DNA Interactions in Cells. J. Vis. Exp. (Pending Publication), e20272, doi: (2023).