08 August 2016 4 2K Report

Hi everyone,

Recently, I am performing IP using anti-Flag Ab(M2, F3165) for Flag-ULK1 overexpression cells. But the efficiency is very low. Even if the input accounts for 1% of the whole cell lysate, the band is significantly higher than the the IP samples for Flag-ULK1.

Note:

1. Flag tag located at N-termial of ULK1, and it contains only one Flag tag rather than three Flag tag.

2. My IP protocol: 20ul Dynabeads protein G mix with 1ul Flag antibody at RT for 40 minutes; the Dynabeads-FLag complex then incubate with the whole cell lysate supernatant for 1 hour at 4 degree. Lastly, the IP samples were washed with ice PBST(0.05% PBST) for 3 times every 5 minutes. The samples were resuspended with 1X loading buffer and subjected to SDS-PAGE and immunoblot analysis. 

So, does anyone has any suggestions for improve the IP efficiency?

Should I try another Flag antibody?

Thanks a lot!

Zhiyuan Li

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