Anti-Acetylated Lysine Western Blot

1.

After the protein transfer, block the mebrane with 3% BSA (do not use milk products) in PBST at room temperature for 60 min.

2.

Wash the membrane with PBST once and incubate with anti-acetylated lysine antibody (0.25-1 μg/mL in 0.1%BSA-PBSt) at room temperature for 120 min.

3.

Wash the membrane with PBST four times and incubate with anti-rabbit IgG HRP (0.1-0.25 μg/mL in 0.1%BSA-PBST) at room temperature for 60 min.

4.

Wash the membrane with PBST four times and add ECL solution for 2 min then expose to the film.

1.

Pre-incubate 10 μg ICP0380 with 40 μL of Protein A beads for 60 min in a 1.5 mL vial, then wash with PBST to remove the unbound antibodies.

2.

After aspiration, add the protein sample (>200 μL in volume in order to avoid surface tension). Incubate the sample with anti-acetylated lysine antibody-Protein A beads at room temperature for 120 min.

3.

Wash the beads with PBST four times. The acetlyated proteins captured in the beads could be either eluted with acid or boiling with SDS-PAGE loading buffer for further analysis.