I have tried a few primary antibodies to detect His-tagged proteins on westerns, and been disappointed with the low signal intensity compared to other primary antibodies used in my lab. I would greatly appreciate hearing about anti-His antibodies that you can recommend I purchase.
My lab uses Mouse monoclonal anti his antibody from Sigma and Goat Anti human/Goat Anti mouse secondary from sigma conjugated with ALP for western. For ELISA we use the same conjugated with HRP..
Good luck..!!
Dear Cote,
We are using Sigma anti-His antibody and it is giving best result as compared to others. I would recommend you to go for only Sigma.
I routinely use abcam ab18184 1:1000 diluted in 5% skim milk/PBST for 1.5-2h followed by secondary antibody, Sigma anti-mouse-HRP for 1-1.5h. The intensity of your His band will be dependent on the efficiency of expression from your plasmid.
Through the years I have tried a number of commercial anti-His antibodies from different hybridomas and my experience mirrors yours: in general their performance is poor compared to what most would call a 'decent' antibody (i.e. you have to use them at relatively low dilutions, etc). Would love to see a systematic comparison between different manufacturers, though.
However, beware that signal intensity, for the same anti-His antibody, may vary widely depending on which specific protein is being probed, even in Western blots (see e.g. PMID 17081490). Perhaps you are dealing with a particularly problematic example?
In our hands, the only one that gave consistent results was Clontech 631212. The only drawback is that it's extremely expensive.
I recently purchased the Clontech 631212 anti-His antibody and it worked better than other. Thanks, Marco, for the excellent suggestion.
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