I treated macrophage cells with LPS (1h to 24h time course) and tried to detect p65 acetylation.
Method 1) I collected total cell lysate with RIPA buffer containing 10mM NAM and 10uM TSA. And then go to western blotting.
Method 2) IP total p65 from total protein lysate (containing NAM and TSA as above) and then blot with Ac-p65 antibody.
But I failed to have Ac-p65 signal. Ac-p65 antibody is from Abcam ab19870, total p65 antibody is sc-372.
Could anybody help? Thank you so much.
You can try Acetyl-NF-kB p65 ( K310) antibody from Cell Signaling (#3045) . The band will always be detected by westernblot.
1) I dont have positive cell lysate from CST now, and in my hands Ac-p65 antibody from CST does not work, that's why I switched to Abcam.
2) Total p65 antibody from SC works well in Western, I am not sure in IP.
3) @Mark, yes, LPS can induce Ac-p65 in macrophages.
Hi Xianfeng Wang,
I know it has been a long time since you asked this question, but I was wondering whether you found a good antibody against acetylated p65 (K310) in the end. Currently, I am struggling to get one that works.
Thank you.
Regards,
Jonas
@Jonas, The antibody from both CST and Abcam work, but in my hand the antibody must be fresh, newly ordered. I don't know why.
@Xianfeng, thanks for the quick reply. Did these antibodies detect endogenous acetylated p65 or had you to overexpress p65?
For endogenous ac-p65, it is really hard for me to detect, but we still can see the band. I just wonder why so many people got very strong band with endogenous ac-p65 in their papers. Sometimes we I emailed people to ask how and I did not receive any response.
It is very easy to get signal once you overexpress p65.
Hi Xianfeng,
I'd like to ask you if you've ever tested other p65 acetylation sites (e.g K218,K221). If yes, I'm wondering if you could recommend some reliable antibodies. I think the antibody you used was against K310Ac. There is a methodology paper published by Chen et al.. They suggested to IP K310Ac first, followed by blotting with total p65 antibody, to get better signals.
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