WB
Western blot analysis of various lysates, using TriMethyl-Histone H3-K27 Rabbit mAb at1:140000 dilution.Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) at 1:10000 dilution.Lysates/proteins: 25μg per lane.Blocking buffer: 3% nonfat dry milk in TBST.Detection: ECL Basic Kit.Exposure time: 90s.
ICC/IF
Immunofluorescence analysis of HeLa cellsusing TriMethyl-Histone H3-K27 Rabbit mAb at dilution of 1:600 (40x lens).Secondary antibody: Cy3-conjugated Goatanti-Rabbit IgG (H+L) at 1:500dilution. Blue: DAPI for nuclear staining.
IHC
Immunohistochemistry analysis of paraffinembedded Human liver using TriMethylHistone H3-K27 Rabbit mAb atdilution of 1:1000 (40x lens). High pressureantigen retrieval performed with 0.01MCitrate buffer (pH 6.0) prior to IHC staining.
ChIP
Chromatin immunoprecipitation analysis of extracts of HeLa cells, using TriMethyl-Histone H3-K27antibody and rabbit IgG.The amount of immunoprecipitated DNA was checked by quantitativePCR. Histogram was constructed by the ratios of the immunoprecipitated DNA to the input.
CUT&Tag
CUT&Tag was performed using the CUT&TagAssay Kit (pAG-Tn5) for Illumina from 10⁵ K562 cells with 1μg TriMethylHistone H3-K27 Rabbit mAb, alongwith a Goat Anti-Rabbit IgG(H+L). TheCUT&Tag results indicate the enrichmentpattern of H3K27Me3 in representative geneloci (MYT1), as shown in figure.