Gel electrophoresis information：10% SDS-PAGE, constant voltage 170V for 60 min

Transfer information：0.45uM nitrocellulose membrane, wet transfer at 250 mA for 60 min.

Lane No. Antigen Loading amount

Primary an-tibody

Primary antibody dilution

ratio

Secondary

antibody dilu-tion ratio

Target band KD Visuali-zation time

1 MCF7 lysate 60 ug

Histone H4 (Acetyle

Lys14) anti-body

1:500 1:2500 ~14KD ECL 1 min

Cell Line: MCF7 cell line

Method of validation: Western Blot

Primary Antibody: Histone H4 (Acetyle Lys14)

Antibody

Secondary Antibody Goat anti rabbit IgG HRP

Dilution ratio: 1:500

Protocol

Treatment of materials: Cells were cultured to 80-90% confluency, and lysed in RIPA buffer.

E.g. Gel electrophoresis: 10% SDS-PAGE, constant voltage 170V for 60 min

Transfer: 0.45uM nitrocellulose membrane, wet transfer at 250 mA for 60 min.

Blocking: 5% milk in TBS for 15 min at RT.

Primary antibody probing: 1/500 diluted in 5% milk in TBS, incubated at 4 C overnight.

Membrane wash: TBS wash 3 times, 5 min/each

Secondary antibody probing: 1/2500 diluted in 5% milk in TBS, incubated at RT for 1 h.

Membrane wash: TBS wash 3 times, 5 min/each

Visualization: ECL 1 min

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