desperate need for an phospho IKK alpha/beta antibody
Julia Scharnert
Friday, 14 August 2009 14:22 UTC
Hi all,
since quite a long time I am desperatly searching for a specific antibody against phospho IKKbeta. I have already tried p-IKKalpha/beta (Ser 180/Ser 181) from Santa Cruz (sc-23470) and p-IKKalpha/beta (Ser 176/180) from Cell Signaling Technology (2697). But I didn´t get any bands at the expected size. Does anyone have an advice concerning a reliable antibody?
Thanks in advance!
Julia
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Replies
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Julia,
The catch to detecting P-IKKb is that you must do an immunoprecipitation. It is possible to detect P-IKKb in total lysates, but it is very difficult. However, if you immunoprecpitiate, the phospho-specific bands become easy to detect. There are two ways to go about this: 1) IP against IKKb – I have tested an assortment of antibodies against IKKb, but most are very ineffective in IP of IKKb (a mouse monoclonal from IMGENEX seems to work the best, but still poor). 2) IP against NEMO – Santa Cruz makes a monoclonal against NEMO that is very good for IP. If you IP NEMO, then just probe for IKKb-P and you should be able to see modification around 10 minutes after TNF or IL-1 stimulation. I use the Cell Signaling antibody against T-loop phospho-IKKb @ 1:1000.
Hope this helps,
-Brock -
Hi Brock,
thanks so much for your prompt and detailed reply! I will follow your advice and try the IP against NEMO first.
Thank you once again!
Julia
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