I used Natural Goat Serum (NGS) for my immunostaining protocol.
However I noticed that NGS is also combined with Bovine Serum Albumine (BSA).
Is there any advantage to use both instead of NGS only?
In your opinion, It is necessary to use both for your staining?
Looking forward to get your feedback.
Dear Angelo Guadagno
I have tried various combinations of NGS and BSA, but it was not useful as it did not yield complete inhibition of non-specific binding.
Use of normal serum is perhaps the gold standard of blocking agents. Usually, 5- 10% normal serum is used. Normal serum carries antibodies that will bind to the non-specific epitopes in your sample, thus blocking your conjugated antibodies from doing the same. This method works especially well if you are working with polyclonal antibodies.
It is important that you do not use normal serum from the primary antibody source, as serum proteins from this source will be recognized by your secondary antibody thereby making your background staining worse, not better. Therefore, it should always come from the same host species as the secondary antibody.
Alternatively, you could use BSA. BSA is used to compete (or block) your antibody from binding to non-specific epitopes in your sample. Your antibodies will not bind to non-specific epitopes any better than these non-specific protein competitors will. Thus, high concentrations of these protein competitors can out-compete your antibodies and lower your background noise. This method is most economical and will often work well for monoclonal antibodies.
If you wish to try, you may use this combination of NGS and BSA in PBS (5% normal goat serum / 0.1% BSA / PBS). Maybe it will help!
Regards,
Malcolm Nobre
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