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 the protocol for chromatin shearing using micrococcal nuclease enzymatic digestion. Chromatin shearing is an essential step in Chromatin immunoprecipitation or ChIp workflow, which helps investigate protein-DNA interaction in human chronic myeloid leukemia cells. In the feature protocol chromatin shearing is performed using sonication.
1. Fixation, Cell Lysis, and Chromatin Shearing
NOTE: Patient samples and Jurkat cells are fixed and lysed with a commercially available ChIP kit according to the manufacturer's instructions with modifications as described previously. The fixation is performed under a laminar flow hood.
Figure 1: Examples of antibody performance in fixed Jurkat cells assessed by SDS-PAGE and Western-Blot. (a) Jurkat cells were fixed for 0 min (bands A and D), 2.5 min (bands B and E), or 5 min (C and F) and the αNFAT2-antibody (clone 7A6) from different manufacturers (bands A-C = manufacturer 1, bands D-F = manufacturer 2) was compared. The antibody of manufacturer 1 showed a better overall performance, binding with the higher affinity even to fixed samples (compare bands A-C and bands D-F). (b) The αNFAT2-antibody (clone D15F1) from a different manufacturer was used. This antibody showed only a poor performance in unfixed samples (band A) and the epitope was masked upon fixation. Therefore, no binding of the antibody could be detected after fixation (bands B and C). Please click here to view a larger version of this figure.
Figure 2: Examples of chromatin of good and poor shearing quality from Jurkat cells assessed by gel electrophoresis. The Jurkat cells were fixed and sheared for different time periods. Fixation was done for 0 min (bands A and D), 2.5 min (bands B and E), or 5 min (C and F). Shearing was performed either for 10 min (bands A-C) or 20 min (bands D-F). Chromatin of good shearing quality is characterized by a DNA fragment size of 200-500 bp which can be detected as a smear in the respective region (bands A and B). Chromatin of poor quality can be recognized either by an almost complete or complete absence of the DNA smear due to an insufficient amount of starting material used (band C) or by a smear in a smaller or larger size region because of inappropriate shearing conditions (bands D-F). Please click here to view a larger version of this figure.
The authors have nothing to disclose.
37 % Formaldehyde p.a., ACS | Roth | 4979.1 | |
1 X PBS | Sigma Aldrich | D8537 | |
1.5 mL tube shaker Themomixer comfort | Eppendorf | 5355 000.011 | Can be substituted with similar instruments |
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 |
DNA LoBind Tube 1.5 mL | eppendorf | 22431021 | |
Halt Protease and Phosphatase Inhibitor Cocktail (100X) | Thermo Scientific | 78440 | |
Small Centrifuge | Thermo Scientific | Heraeus Fresco 17 | |
Density gradient medium Biocoll (Density 1,077 g/ml) | Merck | L 6115 |