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purified antigen and ELISA - non-specific bands in purified antigen for ELISA use (Jan/30/2011 )

Hi everyone! I'm currently doing experiments on vaccine development. And, I'm measuring the antibody titers of our immunized animals. However, I'm having a TOUGH time purifying the proteins that I need for ELISA. I was able to extract the protein that I need but upon doing SDS-PAGE, I found 2 bands on my gel - 1 corresponding to the protein (antigen) that I need and another non-specific band. My question is, would it be okay to use this protein for my ELISA? The non-specific band is very faint so I was thinking if it would affect my ELISA. Another question is would lysed sera have an effect on my ELISA readings?

I'm new to this forum and I am really in need of advice. Thanks in advance!

-BearInTrouble-

It would be simple to try; just coat the protein of interest...you could do both (bands) if you have sufficient quantity. did you not have purified material to create your immunogen?

Lysed sera? Do you mean some of the rbcs lysed in process? You really don't want to use heavily hemolyzed samples.

-sgt4boston-

sgt4boston on Mon Jan 31 11:47:09 2011 said:


It would be simple to try; just coat the protein of interest...you could do both (bands) if you have sufficient quantity. did you not have purified material to create your immunogen?

Lysed sera? Do you mean some of the rbcs lysed in process? You really don't want to use heavily hemolyzed samples.



Thanks Sgt4boston for your reply. I'll try to do as you said. I actually purified the immunogen from E. coli. Then, I added beta-mercaptoethanol in my sonication buffer. After purification with Ni-Sepharose and desalting, I got the 2 bands. As for the lysed sera, yes I think the rbcs were lysed after or during blood extraction. The samples are not actually heavily hemolyzed but I got higher ELISA reading from my pre-vaccination sera compared to the vaccinated sera. :(

-BearInTrouble-