Combine 2 different primary antibodies produced by the same animal - Is it possible to succeed the impossible? (Nov/11/2008 )
I have one rabbit ki67 (proliferation marker) primary antibody and an other one rabbit primary for a neuronal marker. I would like to use them at the same time and try to avoid if possible the mixed up when I will add the anti-rabbit secondary antibody. Is it possible to succeed the impossible?
Many Thanks
It is possible to use them together with conventional western systems, so long as the proteins are different sizes. If you are doing something like immuno fluorescence, you will have to add one primary, detect using one colour secondary and the do the second, detecting with another colour.
For WB, you can do that if: the proteins are of different sizes; both the primary Abs detect clean, single band. I would rather cut the membrane into several pieces if the proteins are separated enough than mix Abs.
For IF or anything similar: I am afraid that I don't agree with this option. The chance of co-localization due to 2nd Ab detection is very high. If you have to do it, then conjugate the two Abs with different colour and use direct staining instead of 2nd Ab.
I second this suggestion. Following transfer, stain your membrane with Ponceau to determine where your proteins of interest should be based on their MW, cut the membrane accordingly to have your proteins of interest on two different membranes, then blot them separately.
Many Thanks
in IHC/ICC: not possible,
for IB: only if differently sized targets and no unspecific background is given...
See Immunoportal.com
Three mouse monoclonals:
Four mouse monoclonals:
Three mouse monoclonals:
Two mouse monoclonals:
I am not sure if it is possible for polyclonal antibodies. I usually combine rabbit polyclonal with mouse monoclonal for IF and secondary antobodies labeled with Alexa fluor 568 and 488. But the intencity of the fluorescence was weaker compared to the single staining.
In IF it has to be antibodies of different species or different subclass to avoid cross reactions. In enzyme based immunohistochemistry this does not matter, two or more abs from same species/subclass can be used. Enzyme based immunohistochemistry should not be used when coexpression is expected!
We did this by Zenon labelling one antibody. It worked beautifully!!