WB
Western blot analysis of Histone H3 (acetyl K23) on different lysates with Rabbit anti-Histone H3 (acetyl K23) antibody at 1/1,000 dilution.
Lane 1: HeLa cell lysate,
Lane 2: HeLa treated with 500 ng/mL TSA for 4 hours cell lysate,
Lane 3: NIH/3T3 cell lysate,
Lane 4: NIH/3T3 treated with 500 ng/mL TSA for 4 hours cell lysate,
Lysates/proteins at 40 µg/Lane.
Exposure time: 1 minute;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.
ICC/IF
Immunocytochemistry analysis of HeLa cells treated with or without 500 ng/mL TSA for 4 hours labeling Histone H3 (acetyl K23) with Rabbit anti-Histone H3 (acetyl K23) antibody at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-Histone H3 (acetyl K23) antibody at 1/100 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (488) was used as the secondary antibody at 1/1,000 dilution. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (594) was used as the secondary antibody at 1/1,000 dilution.