I've grown different cells wanting to assess caspase-8 levels in the whole cell lysate. I haven't treated them with anything. I'm unable to visualize caspase-8 in a western blot. What might be wrong? do i need to treat the cells to visualize caspase-8?
Depends on your antibody. If you have an antibody that can only interact with cleaved caspase 8 then you will have to induce caspase 8 activation with an appropriate inducer
I agree with Gaurav. Make sure your antibody is for detecting total pro-caspase 8. If the antibody is for cleaved caspase 8, then you will only detect it in cells that have been stressed.
We like the BD antibody for this application. They have an antibody that recognizes the prodomain (DEDs) that blots fairly well. It will recognize everything the activation specific antibody does not (but will not recognized the active catalytic domain).
Thank you all for your suggestions. the antibody i'm using is from cell signaling and on their product page, it says it can detect both cleaved (43,18 KDa) and non-cleaved (57 KDa). i've used the same antibody before on treated cells and got a band at 57, 43 and 18 KDa, so i know the antibody works. i just cannot figure out as to why the same does not work for non-treated cells. i'm repeating the experiment with a borrowed antibody to see if the situation changes.
once again, thank you all for your inputs.
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