Hello!
This is my first foray into immunoprecipitation. I'm IPing HER2 after a treatment with a chemical. I know from previous westerns(total protein) of the same treatment that the HER2 receptor is phosphorylated upon these treatments.
My goal with the IP was to prove that downstream effects i'm seeing is via the HER2 receptor.
I was able to IP HER2 as I ran a western and saw HER2 expression but the expression increased with treatment which I did not see with blots run on total protein samples. What could be the reason for that?
Also, I was unable to detect any phosphorylation of the HER2 receptor with anti-phospho HER2 antibodies. I don't understand why as I know the receptor is activated upon treatment.
I used CST monoclonal HER2 antibody validated for IP.
Any help is much appreciated.
Thank you.
Hi! Sarah,
I’m using the same antibody of HER2 for IP and later western blot. It’s source is rabbit. The secondary is also rabbit. Does that help?
Hi! Sarah,
I’m eluting the antigen off of the antibody-bead complex and running them on a gel.
Thanks Sarah.
Would my best bet then be to use a differnt species of the antibody to detect my target via western blot after IP?
Sarah Votah explained pretty well and covered most. I would suggest run Input with and without treatment along with IP samples. Also, make sure to use appropriate concentration of antibody for your IP experiments.
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