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.
Protocol
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. Prepare a 1.5 mL tube with 1 mL of 37% formaldehyde, a 1.5 mL tube with 1 mL of 1.25 M glycine, a 5 mL tube with 4 mL of 1x PBS and a box with ice for the fixat…
Representative Results
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 o…
Divulgaciones
The authors have nothing to disclose.
Materials
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)