SDS-PAGE protein analysis with antibodies alone (not WB) - (Apr/23/2015 )
My partner and I are doing a protein detection via 2D SDS-Page for the CagA protein from H. pylori, we have a primary and a secondary antibody (fluorescent) specific for CagA. Our problem is that our university lab does not have the supplies to run a Western Blot. So we were looking to see, if any one might suggest a protocol or tips, on directly exposing our gels to our antibodies in an attempt to detect through that method?
Thank you for any advice you might be able to provide.
we've done it in the past.
to ensure that the proteins don't elute during incubations you have to "fix" the proteins more permanently than with acetic acid (in methanol or ethanol). we used tca.
wash out sds with ethanol (10%)
incubate with primary, wash thoroughly (you're not just washing a surface, you have to remove unbound antibodies from inside the gel).
then secondary and more washes.
keep in mind that most of the protein you will see is at or near the surface of the gel, certainly not all that was applied to the gel. also, the longer you allow the antibodies to impregnate the gel, the more and longer you have to wash.
all in all, it's a lot simpler to transfer to membrane.
by the way, now that i think of it, you can perform a capillary transfer to a membrane (if you have any or can acquire some from another lab) nearly as effectively as electrophoretic transfer and won't require any equipment which you don't already have in your lab (probably).