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high background with acetyl-lysine antibodies for IP immunoblot


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#1 crom80

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Posted 07 February 2014 - 08:06 AM

Hi

 

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??

 

Thank you.



#2 lilin_hust

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Posted 09 March 2014 - 01:21 PM

I did that once and am also tryiny to do another one.

 

When you use POI specific antibody, the band is clear, this means you get the protein, but it is possible there are other endogeneous acetylated proteins pulled down also, so when you use anti-acetyl antibody, you may see many bands (your POI as well as those potential endogenous acetylated proteins).

 

So I suggest you don't care too much about other bands, as long as you are quite sure which band is your POI.

 

Hope this helps, and we can further discuss on this topic :)






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