I am going to block receptor with specific antibody at 37 degrees for 24 hours and then going to treat my cells with stimulant. Will that work for blocking receptor?
If your antibody binds to ligand binding area/s (epitope) of the receptor and compete for the ligand/s, there may be good chance for successful blocking. If you are in search of new blocking antibody, polyclonal antibodies are better choice than monoclonals.
If the stimulant and the antibody target the same receptor, within the same general locality on the receptor, your experiment MAY work. Additional concerns to address will be 1) recycling of the receptor/ production of fresh receptor moiety; 2) internalization of receptor upon antibody binding if it does occur, which may have a secondary effect altogether;3) Possible signalling by the antibody binding to the receptor. Think about these when you design your experiment. Literature review should help you work around at least some or all of these concerns.
yes,sanath actually these were my main concerns and thats why I wanted to ak for time of preincubation. In 24 hours, possibility of above concern are alot. Do you have any suggestions for incubation time?
Thanks Sanath and Kameswara.
There is actually no way of telling if these will affect your system or not. Look into the previous literature for similar experiments or optimize the process yourself.
I agree with Rodrigo. Antibodies to cell surface receptors are often internalized and degraded. A 1 hr incubation with the antibodies should be enough. You should also have an antibody only control in the experiment.
Yes, that should be helpful. I am planning to use 5 ug/mI antibody for 3 hours. What do you think about this incubation?
And I am going to block lipoprotein receptors. And yes, I am using antibody control as well.
Thanks Rodrigo and Steingrimur.
Hi Monica. I'm quite familiar with lipoprotein receptors. We usually used >100 ug/ml of polyclonal antibodies to LRP-2/LRP-2 and added them 30 min prior to the assay. We used polyclonals because those receptors are multivalent.
Forgot to add the papers
Article gp330 on type II pneumocytes mediates endocytosis leading to...
But Stefansson, I have one doubt that 100 ug/ml is alot of antibody. Can I use 10 ug/ml instead?
Hi, I think the antibody inhibition depends on whether the LDLR you are looking at binds more than 1 copy of the ligand. We almost never got very robust inhibition of the LRP-1/LRP-2 receptors with polyclonal antibodies. The exception was inhibition of a2M:proteinase complex, which binds to 1 site on LRP-1.
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