Protein G - (May/22/2007 )

Hi there!

I have a brief questions. I read that you can use Protein G as a kind of "secondary antibody" to detect proteins in western blot. It recognizes IgG Fc. My questions in now: if i want to use 2 different primary antibodys from different species (eg. mouse and sheep) against different proteins (lets say my protein of interest and actin) could i use both primarys in on reaction and then detect both of them with protein G simultanousely? That would save stripping and reprobing my westerns...

Does anyone have experience with that?

Stardust

Hi Stardust,

Unfortunately I`ve got some experience with Protein-G-Hrp (BioRad). It is a grand idea to detect both abs at the same time, but I had very bad experience with proteinG. It gave me tons of bands and the backround was quite strong, whereas with the proper 2n ab, no background and stong signal. So, you have to test your system and probably spend a lot of time optimising it. I think it is worth the effort to try, but if you`ve got a nice system for both 2nd abs and stripping works fine, I would stick to it.
On the other hand, if you get good and single signals for both abs, you could try and detect them at the same time incubating the blot with both 2nd abs subsequently (I did it once and it worked, but since then I haven`t got the courage to try it again.). It can generate a lot of problems and nonspecific signals, though, so I would only consider it in a desperate need.

Does Protein-G-HRP work fine for you? Which brand are you using?