Frequently Asked Questions

Yes, there are two options:

1. First of all, theoretically this should not be a problem when the antibodies (Ab) used for IP and WB are from different species (e.g. rabbit Ab for IP and mouse Ab for WB; detection with a labelled ani-mouse antibody that does not react with rabbit IgG heavy chains). However, as a matter of fact, some secondary Abs may x-react with IgG of other species, resulting in a residual reactivity with the abundant heavy chains. Therefore, you should take care to select a perfectly pre-absorbed secondary antibody.
2. If both IP and WB are done with (the same or different) rabbit Ab(s), you may use labelled protein A instead of a anti-rabbit Ab for detection. In our hands ¹²⁵I labelled protein A  proved absolutely non-reactive with Ab heavy chains separated by SDS PAGE. Protein A labelled by peroxidase should work as well. However, there are some reports that certain brands or lots may show some residual reactivity with denatured heavy chains.