Histone H3 (acetyl K4) Recombinant Rabbit Monoclonal Antibody [PSH03-71] - BSA and Azide free

☑ Cell treatment (CT)

Western blot analysis of Histone H3 (acetyl K4) on different lysates with Rabbit anti-Histone H3 (acetyl K4) antibody (HA750905) at 1/1,000 dilution and competitor's antibody at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa treated with 500ng/mL TSA for 4 hours cell lysate
Lane 3: NIH/3T3 cell lysate
Lane 4: NIH/3T3 treated with 400nM TSA for 18 hours cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 15 kDa
Observed band size: 15 kDa

Exposure time: 1 minute 22 seconds; ECL: K1801;

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 (HA750905) at 1/1,000 dilution and competitor's antibody at 1/1,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Cell treatment (CT)

Immunocytochemistry analysis of HeLa cells treated with or without 500ng/mL TSA for 4 hours labeling Histone H3 (acetyl K4) with Rabbit anti-Histone H3 (acetyl K4) antibody (HA750905) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Histone H3 (acetyl K4) antibody (HA750905) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

☑ Cell treatment (CT)

Immunocytochemistry analysis of C6 cells treated with or without 500ng/mL TSA for 6 hours labeling Histone H3 (acetyl K4) with Rabbit anti-Histone H3 (acetyl K4) antibody (HA750905) at 1/1,000 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Histone H3 (acetyl K4) antibody (HA750905) at 1/1,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated 500ng/mL TSA for 4 hours with Histone H3 (acetyl K4) (HA750905) or Normal Rabbit IgG according to the ChIP protocol. The enriched DNA was quantified by real-time PCR using indicated primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Dot blot analysis of Histone H3 (acetyl K4) on different proteins with Rabbit anti-Histone H3 (acetyl K4) antibody (HA750905) at 1/1,000 dilution. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution for 1 hour at room temperature.

Lane 1: Unmodified Histone H3 (negative)
Lane 2: Mono-Methyl-Histone H3 (Lys4) (negative)
Lane 3: Di-Methyl-Histone H3 (Lys4) (negative)
Lane 4: Tri-Methyl-Histone H3 (Lys4) (negative)
Lane 5: Acetyl-Histone H3 (Lys4) (positive)
Lane 6: Acetyl-Histone H3 (Lys9) (negative)
Lane 7: Acetyl-Histone H3 (Lys27) (negative)

Proteins loading: 100ng, 25ng, 5ng;

Blocking and dilution buffer: 5% NFDM/TBST;

Exposure time: 50 seconds.