Hello,
My question pertains to determining surface protein concentration. As for the protein in mind, it must be total surface protein concentration, not just a specific one. The problem is that we can't use ELISA or anything that uses a specific antigen-antibody interaction because we don't know the surface proteins expressed, and over time we are using multiple different strains of bacteria to produce different antigens.
We currently use BCA to determine concentration, which works sometimes, and sometimes gives a very non-linear relationship compared to the standard curve.
Is anyone else aware of a test we can use that doesn't require the specific production of associated antibodies?
Thank you,
Dan
You can use flow cytometer for surface antigens.
Aloso, use of Bradford for protein estimation is too better than BCA.
Dear Masood,
Thank you for your response. The only problem with the 2 suggestions you have is:
1) For flow cytometry, you must know the composition of the protein on the surface in order to attach the fluorescent molecule (must know the protein expressed, which is different for every strain of bacteria)
2) Bradford absorbs at 600 nm, which is also the wavelength that our bacteria absorb at, which wouldn't allow us to differentiate between the two.
Thanks again
D
Cell surface biotinylation is the classic approach here. Treat cell with a membrane-impermeant reagent that will introduce a biotin tag onto cell surface proteins. Make a cell lysate and "pull out" biotinylated proteins using streptavidin resin. Analyse for the presence of your biotinylated protein on an immunoblot of a sample of the lyate before and after treatemnt with the streptavidin resin. There are hundreads of articles usign this technique but a recent review is: Elia, G., Biotinylation reagents for the study of cell surface proteins. Proteomics, 2008. 8(19): p. 4012-24.
What do you mean by "cell surface"? Are you specifically looking for proteins anchored in the outer periplasmic membrane? In the periplasm? In the inner membrane? Something else? Your methodology will differ greatly for each of these targets.
Also, your BCA works at 562 nm. Bacteria "absorb" (or more accurately scatter) there too, even more strongly than at 600. If you're concerned that this might interfere, spin the bacteria down, and measure the supernatant.
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