If I am doing an IP for a mouse and a human protein with an antibody that is raised in Rabbit and recognizes both the mouse and human protein, and later when I want to do WB, is it okay if the host of the primary WB antibody is also Rabbit? I know that to avoid cross-reactivity, the host (e.g. rabbit) of the WB primary antibody should be different than the host (e.g. mouse) of the target protein. But can the IP antibody and primary WB antibody be from the same host?
Hi,
If the target protein has the molecular weight far from Ig heavy chain (50 kDa), it should be ok to use Abs of the same host in IP and WB. Otherwise, don't do it. The overpowered signal from the IP heavy chain will cover the signal from the target protein in WB. Good luck for your experiments :^)
Hi,
the protein you IPed should not be in the range of 50kd (heavy chain) nor 25kd (light chain); you can cut out these regions from your filter, or use loading buffer without betamercapto (if you know where your prot will run in this condition) then the Ig will run at 150kd (however expect some 50 and 25kd bands)...there is also kits to link the IP antibody to beads so you don't get the antibody on your western blot... in any cases do not expect miracles!!! better spend monay on another primary antibody (mouse monoclonal?)