Nuclear lysates were prepared as previously described31 (link). Nuclear extracts were separated on either 8%, 10% or 20% SDS-PAGE gels. Blots were probed with primary antibodies (H2A-Ub[K119] [Cell Signaling Technology], H2A [Cell Signaling Technology], H2B-ub [EMD Millipore], H2B [Cell Signaling Technology #12364, clone D2H6], H3 [Cell Signaling Technology], H4 [Cell Signaling Technology], HA [Cell Signaling Technology #2367, clone 6E2], TRIM37 [Abcam ab95997, or custom made by 21st Century Biochemicals against a synthetic peptide corresponding to amino acids 444–460 of the human protein followed by affinity purification] or], RNF2 [Abcam ab28629], and α-tubulin [in-house]) overnight at 4°C, washed five times in TBS plus 0.1% Tween (TBST) and then incubated with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. Membranes were washed five times in TBST and visualized on autoradiography film after incubating with ECL reagent (Supersignal West Pico or Supersignal West Femto; Thermo Scientific). Immunoblots were quantified using Image J software version 1.47v (NIH).
Protocol detail
Immunoblot Analysis of Histone Modifications
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Affinity purification
Amino acids
Antibodies
Antibody
Autoradiography
Clone
Gels
Human protein
Immunoblots
Membranes
Peptide
Rnf2
Sds page
Trim37
Tween
α tubulin
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Variable analysis
independent variables
- SDS-PAGE gel concentration (8%, 10%, or 20%)
- Levels of histone proteins (H2A-Ub[K119], H2A, H2B-ub, H2B, H3, H4, HA, TRIM37, RNF2, α-tubulin)
- Nuclear lysates were prepared as previously described (reference to a previous publication)
- Blots were probed with primary antibodies overnight at 4°C
- Blots were washed five times in TBST and then incubated with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature
- Membranes were washed five times in TBST and visualized on autoradiography film after incubating with ECL reagent
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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