I am currently examining the acetylation of a protein of interest with or without a drug (HAT inhibitor)
I pulldown POI with specific antibody (rabbit) and run SDS gel.
When membrane is incubated with POI specific AB I see the band clearely (no bands in IgG ctrl) and when I blot for beta actin I see that only total lysates show band whereas IP lysate samples do not suggesting IP for POI worked.
When I incubate with acetyl-lysine specific AB (i tried both rabbit and mouse) I see multiple bands not specific to POI (acetyl-lysine incubation was done on separate duplicate membrane not incubated with POI or actin AB)
During my initial attempt I had blocked in milk and saw multiple bands with rabbit acetyl-lysine. so I ordered a mouse and while waiting for AB I blocked the same membrane in BSA and reprobed with the same rabbit AB used previously.
Next day the blot looked amazingly clear for acetyl-lysine bands. When mouse AB arrived and tried, the same clear bands showed up.
Now I am trying to repeat with a different drug compound and IP worked when POI and actin were probed.
The duplicate membrane was blocked in BSA (skipped milk block since initial attempt show multiple bands I assumed milk was bad blocker) and incubated with mouse acetyl-lysine AB.
I saw multiple bands again which I don't think is due to pull down AB since from different host. I tried blocking in BSA again and incubated with rabbit acetyl-lysine AB and saw same bultiple bands.
I am planning to try blocking in milk and reprobing.
Do any acetyl-lysine IP probing experts have suggestions??