I have acquired PSA-specific IgG1 antibody, which is biotinylated on the Fc region and we want to use it as primary Ab in immunodetection on western blot. We have secondary Ab conjugated with reporter protein, which binds to Fc region of IgG1, so I am wondering if biotin tag would perturb binding of secondary Ab to the constant region of the primary Ab? I have read that there are 2,5 biotin molecules per Ab in average, so having in mind biotin fairly low molecular mass (240 Da), there shouldnt be any problems. If anyone had similiar doubts, please let me know. Thanks.
Use a streptavidin-enzyme conjugate as the secondary. That's the purpose of the biotinylation of the primary.
Your secondary antibody may still work, but probably with lower efficiency than it would with an unbiotinylated primary antibody. You may need to use a higher concentration, and this will contribute to a higher background.
I think the secondary polyclonal antibody should generally work in cases like this one, but using the streptavidin conjugate is of course a good option.
Of course, I know that streptavidin conjugate would work the best, but my options are limited, so I have to try this work with current material at my disposal. Than you both for your answers.
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