Will the TrxA tag in the E. coli prokaryotic expression vector pET32a cause non-specific binding in WB or ELISA experiments?
I used a fusion protein with TrxA to coat an elisa plate to test whether there were antibodies in the blood. There was a big difference between the two blood samples. After the serum was diluted 800 times, one had an OD value of 1.2 and the other had an OD value of only 0.15. They were temporarily determined to be positive and negative. . But I used these two blood samples for WB primary antibody, and the results showed that the negative WB also had bands.I am now confused whether the fusion protein is too non-specific or whether the negative ELISA result is actually positive instead of negative.
The background signal of Trx with your antibodies can be checked if you coat with Trx only and measure the signal.
Although, it seems more likely that you have the problem that ELISA and Western Blot are not necessarily identical assays, so you could get signal in Western Blot but not ELISA for various reasons, e.g. differences in binding native and denatured target protein. Your fusion protein could also bind the plate surface in a preferred orientation like the hydrophilic Trx towards the solution, so accessibility to the target protein may be limited or less epitopes available for binding.
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