ELISA protocol - (Mar/05/2008 )

Hello,
I'm a newbie in ELISA.
My protein isn't detected with a western blot and I hope ELISA is more sensitive. The problem is that my protein is intracellular so I guess I have to lyse my cells before doing the ELISA but I don't know which reactant I can use for that (that won't interact with the detection..).
If you have a good protocol for ELISA, I'm also interested. I found some in papers but they're not always detailled so It's difficult.
Thanks a lot

What cells are you working on and what protein? What cell compartment is it in?

To lyse cells you can use mechanical means: French press, (might be harsh, though) or sonication on ice. Or you could use an enzymatic method (lysozyme).

Tougher cells (yeast) need harsher techniques. You are right to think about risks to your target protein. Is it globular (probably more robust) or fibrous (exposed along its length). Is it a glycoprotein (might affect folding and packaging such that in ripping open the cell you access the protein in a non-physiological form).

Thanks for your answers

I use cells infected with a virus so not all cells in my population are infected and my protein is producted in a very low level by the virus, that's why I can't see it in a western blot but I hope an ELISA would be more sensitive.
I would like to infect HeLa and Jurkat. I think total cellular extract would be the best, and I was thinking to do it with a buffer (the same principle that's used for the cell lysis when you work a luciferase assay) but I don't know if it's compatible

for a direct elisa:

1) coat plate with sample (in pbs, tbs or carbonate buffer)

2) block with appropriate blocking agent (bsa, normal serum from source of secondary antibody)

3) incubate with primary antibody

4) wash (3x) with pbst or tbst (.05-.1% tween-20)

5) incubate with secondary antibody conjugated with hrp or ap (do not use phosphate buffers if you use ap conjugated secondary antibody)

6) wash

7) incubate with appropriate substrate

8) read results