Western Blot - (Apr/14/2005 )

Hello everyone,
I am doing western blot using a recombinant protein. I transfer the
protein to nitrocellulose membrane and confirm by Ponceau-S staining.
The secondary antibody is HRP mouse anti-human IgG1 (1:5000).I don´t know why my secondary antibody, recognize all the protein. I am thinking about using the HRP goat anti-human IgG1 instead of mouse, because when I use goat anti-human IgG my western blot works. What do you think about this?

Thanks in advance,

HI!!

Wath do you use to incubate your menbrane? Is it gloria or something like that? One day, I had a big problem of aspecificity and when I remplaced gloria by Amersham "Milk", this problem was resolved. More, before primary Ab was 1:1000 and secondary Ab 1:100.000, with Amersham it became 1: 5000 for the 1 Ab and 1:250.000 for the secondary.

And now, have you always problem with your western blot?

Hope to help you,
Ciao