Use of carbonate buffer to dilute antibody?
Posted 12 October 2010 - 11:55 AM
Is it ok to use BSA?
Posted 12 October 2010 - 12:05 PM
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Posted 13 October 2010 - 12:22 AM
Is it necessary to use a carbonate type buffer for capture antibody dilution. Is it ok to use BSA?
The coating buffers most used are 50 mM carbonate, pH 9.6; 20 mM Tris-HCl, pH 8.5; and 10 mM phosphate-buffered saline (PBS), pH 7.2 . I don't think you can use BSA to dilute capture antibody.
You may use it to reduce high background when using polyclonal antibodies (PCA)as capture antibody. Polyclonal serum will result in strong signal, and in this case you may use BSA to preabsorb the serum using competitor protein such as BSA. In ElISA, you can use BSA as blocking agent because of its ability to compete with nonspecific factors in the test sample for available plastic sites.
Hope this helps.
Posted 13 October 2010 - 12:58 AM
I've always used either carbonate buffer pH9.6, or PBS to coat ELISA plates. Just dilute you coating antibody directly in this buffer and add to the wells.
Posted 13 October 2010 - 04:16 AM
Use BSA or other proteins for blocking uncoated plastic surfaces. there are also commercial blockers which you can try. if you wish to keep your plates dry and for extended periods...use some sucrose in buffer as final block step decant and allow plates to dry inverted. then keep dry in plastic bag with dessicant and refrigerate.