I want to know primary and secondary antibody concentration/amount for western blot experiment. What factors are responsible for determining antibody amount? Thanks
Ideally, monoclonal primary and secondary antibodies should be 1:3000 and 1:5000 respectively for western blot analysis.
Thanks Antoni for your suggestions. Thanks Rishabh. All companies do not provide antibodies with same concentration. In this case, dilution should not be same. That's why I asked about final concentration. Thanks once again.
Antoni is correct. However, as long as there are undefined formulated antibodies on the market, such as serum, culture media, ascetic fluid, the proper dilution may change every time when you buy the same product yet again. Only antibodies that come in defined mg/ml purified IgG can be diluted to its optimal concentration. Beware though, that with polyclonal antibodies each animal will generate a different product with a different optimal dilution. And there is a big difference between proteinA-purified polyclonal antibodies and antigen-affinity purified polyclonal antibodies and between monoclonal antibodies derived from a hybridoma clone and recombinant monoclonal antibodies.
Hi Kartik,
It is best to run a serial dilution with the primary antibody in order to determine the optimal dilution needed for your assay. I would suggest starting with dilutions of 1:500, 1:1000, 1:2000 and then determine if you need to increase or decrease the dilution based on the signal you get with each of the recommended dilutions. As Jan suggested there are various from of antibody so it is important to distinguish between different "purified" forms of antibodies to ensure they are indeed purified and not just filtered serum. Another consideration is often companies will purify different lots of the same catalog # antibody from different bleeds of the animal which has drastic effects on the quality and concentration of the end-product. To learn more about our company's initiative to ensure reproducibility with our antibodies please check our our website, https://www.phosphosolutions.com/reproducibility/
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