I´m haveing trouble with some samples I´m trying to determine Serpina 5 content by ELISA assay. I´m using a comercial strip kit.
With my director we are starting to think the protein is forming some kind of agregate that is blocking the recognition site for the antibodye or something like that, so we were thinking of doing a cracking step, like in denaturing electroforesis, to the samples before diluting for the assay.
Is this posible? has someone done it and can share your experience?
Thanks a lot for any help you can give!
Hello Pau,
I don't think is a good idea, in some cases, the antibodies recognize conformational epitopes that could be affected if you crack the samples. If your concern is potential aggregates I will try to dilute the samples in a different buffer first and try a mechanical disruption of the aggregates.
Depending on much you know about the structure and sequence of your antigen, you could try changing pH, adding EDTA (to complex bivalent cations), changing salinity or add chaotropic/denaturing agents (MgCl2, NaI, guanidinium salts, urea).
You might need to test a lot of conditions and possibly remove the added chemicals before you add the sample to your ELISA.
Could you quantify per blot ? If so, your epitope is stable in SDS buffers. But your detection antibody might not like... ! A simple dot blot of SDS samples in serial dilutions might help, the washing and blocking steps eliminating the detergent. Another way is to supplement the EIA dilution buffer by soft hofmeister series chaotropic agent like NaSCN.
Thanks a Lot Laurent!
We dont have the antibody for blotting yet, so we cant know this. The previous studies were SDS-PAGE, mass spectrometry and SEC. But we wanted to confirm by ELISA and maybe use this technic as a routin follow.
Thanks everyone!
We´ve tryed changeing the buffer by diafiltration, but not tested yet EDTA on that condition. I will try that next.
The SDS/b-mercaptoetanol is a little to harsh I think, but was sugested by someone at the lab and it gives me no good results so far.
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