Far western blot: probe concentration - (Feb/12/2013 )

Hello

I want to do far western blot with my his-tagged protein as the probe. The size of my protein is 27 KDa. But I am not sure how much protein to use as probe? If anyone could please suggest the concentration of probe and the subsequent concentrations of the primary and secondary antibodies that would be great.

Thanks

Bioscientist

I'd suggest spotting the original sample on membranes and then testing binding and detection without first doing a western. It's much easier to do detection optimization without having to do gels.
If you do serial dilutions, this will also provide information about the sensitivity of the assay.