Secondary Antibody Choice - (Oct/16/2006 )

Hi everyone,

the manufacture of my primary (polyclonal goat antibody) recommends using donkey anti-goat IgG-HRP as a secondary antibody , however i got lots of nonspecific bands when i used it and the band i was looking for was very weak.

when i used donkey anti-mous IgG HRP I was able to get much cleaner blot and very strong band at the expected molecular weight.

It doesnt make sense to me but did this happen to anyone of you before.

thanks a bunch

And this was when using the same primary antibody with both the anti goat and anti mouse secondaries?

interesting, this could interest a friend of mine.
I borrowed him a few microliter of antigoat from santacruz. It worked perfectly. unfortunately it's an old batch from 2001, and when he ordered two new batches he got several bands.
Maybe I should suggest him to use an anti-mouse?!
Did you try with other goat antibodies?

I think this is probably just a non-specific interaction. But I could be wrong. Does your secondary data sheet for your anti mouse say that it should not react with goat?

He Missle

I didnt try it with other goat antibody. Im worried it might be unsepcific interaction But i tried it with a positive control (cell lysate i bought from the company and recommended as positive control for this primary Ab) and i got the band at the expected molecular weight.

If your secondary antibody does not specifically say that it has been purified to not react with other species, it is possible (I think) that the mouse serum has natural goat antibodies in it that would react with your goat primary.