Hello everyone, I am trying to detect HBx protein from Hepatitis B Virus through Western Blot. Knowing that the molecluar size of this protein is 17kD can anyone give me an indication about the protein concentration that I should use to get a good detection. Thank you in advance.
Dear Iskander,
Multiple variables go into this, including sample preparation (are you running purified HBx protein or lysates of some sort) and primary antibody (depending on strength of your antibody, you may need to use more or less). It never hurts to run a titration of protein. For example, if using cell lysates, you could try 40ug, 20ug, 10ug, and 5ug side by side on a western blot. Then, if you don't have a known antibody concentration, a good starting point is 1ug/ml. If there are too many background bands, you can reduce this concentration (though background can come from secondary antibody too!). There isn't usually a single "magic" number of protein or antibody...unfortunately, it usually needs to be adapted for different assays!
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