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

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Citar este artículo
Chromatin Immunoprecipitation Assay: A Technique to Study Protein-DNA Interactions in Cells. J. Vis. Exp. (Pending Publication), e20272, doi: (2023).

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