I need help removing IgG from protein homogenates!
tguettou at newssun
Sat Jan 11 14:39:44 EST 1997
On Thu, 9 Jan 1997, Jil Tardiff wrote:
> Hi folks,
> I could really use a hand with a confounding problem.
> I have some transgenic mice and I am trying to study the transgenic
> protein (simple). The problem is that our animal facility has become
> infected with *something* of late and the end result is that my mice are
> really making boatloads of IgG. Thus, whenever I perform a protein
> determination my results are skewed because of the IgG load. What's
> more, when I run an SDS-PAGE the Tg proteins are not running correctly.
> The subsequent Western (since we're using goat anti-mouse HRP with ECL
> to detect) has a whopping IgG H+L signal (detectable without primary of
> course) that is making a real shambles of my attempts to characterize
> the mutant protein. The primary Ab is hybridoma supt of c-myc (
as mentioned before, to remove your IgG you can use either Protein G or
Protein A depending on the IgG subclass. It is very easy to determine
which subclass you have and most companies offer information what
subclass binds best to protein A or G.
To get rid of your problem with the Western ( secondary recognizes
Heavy/light chain), you can crosslink Biotin to your primary and use
Streptavidin-HRP(there are some kits available). If you want to increase
your sensitivity you could also use a Streptavidin bridge and use Biotin HRP.
This should take care of your problem.
Hope that helps.
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