reducing background in western... - ...by blocking the antibodies (Oct/09/2006 )

Hi all!
I extracted a protein directly from a SDS-PAGE gel band and use it to inoculate a rabbit. As we expected, the antibodies yielded a lot of inespecific bands in western blot. I´m looking for the best way to reduce the extra bands. For example, I´ve tried to block the antibodies with coli extract but it wasn´t so good (I added the extract to the milk-PBS buffer + antibodies). Is it better to block the antibodies directly in the serum? Is it preferable to go ahead with antibody purification? (but those protocols are a PIA!).
So, does anybody have a good protocol for blocking polyclonal antibodies?? Thanks a lot!!

In my experience you will always see non-specific bands when using serum. You could dilute it way out to try and make it better, but as far as I know purification is the only way to increase your specific signal while decreasing or eliminating your non-specific bands.

hi there,

you best bet would be to purify your antiserum by immunoadsorption against your antigen. there are a number of ways to do this. this method will give you the cleanest results.

other methods of IgG purification such as protein G chromatography, ammonium sulfate fractionation, or affigel blue chromatography will partially purify IgG's, but will not do so with the specificity that you might need.

if you'd like an immunoadsorption protocol or two to try, email me at jonmike.reed@gmail.com