Tips on using Furin - (Nov/14/2007 )
Has anyone used Furin to digest proteins? The buffer contains BME and Triton and that seems to messing up our system. I m using Furin treated ELISA plates for my experiment and the antibody binding decreased instead of the expected increase.
Does anyone know if I can vary the buffer system: it contains:
HEPES 100mM, 0.5% Triton X-100, 1mM CaCl2, 1mM BME.
I wonder what will happen if you treat the protein with furin first, coat the plate with the degraded protein then test for antibody binding?
We use precoated ELISA plates, and Furin treat them in lab before proceeding to the actual experiment, this the first time we are doing it and the company protocol/representative has no idea either. So what we are thinking is happening is that the BME in the buffer may be denaturing the protein and then when we add the antibody we get nothing, ppbly since the conformation of the antigen on plate changed due to BME.
So what i m looking for is a buffer that can be used for this enzyme that excludes or substitutes the denaturing agents in the buffer, if anyone has tried it.