I am trying to detect an mRuby-tagged fusion protein on a western blot. Could someone please recommend a good antibody for detecting mRuby on a western blot? I would really appreciate your input. Thanks!
It seems a little bit strange to me, to detect a fluorescent protein with a fluorescently labeled antibody. Did you consider a native PAGE?
http://www.assay-protocol.com/index.php?page=diverse-native-PAGE
http://www.assay-protocol.com/molecular-biology/electrophoresis/native-page
Hi Michael,
Thanks for your suggestion. Maybe my question was not very clear. I was wondering if anyone had used an antibody such as an anti-RFP antibody that might recognize a mRuby-tagged protein on a western blot. Since we want to compare the relative abundance of an mRuby-tagged protein between different samples, we want to perform a western blot for extracts from the different samples. Unfortunately immunofluorescence is not as quantitative as western blotting. So, we prefer performing a western blot in this case.
Hi Jyoti,
What kind of detection are you using for your western blots?
Also, since our sample is a complex whole tissue extract containing a mixture of proteins and we don't have an antibody against the protein that is tagged with mRuby, unfortunately, we cannot perform a native PAGE or native PAGE followed by western blotting.
There are antibodies to “RFP-tag” which is the same species family, both RFP-tag and mRuby1/2/3 being derived from eqFP611. There are monoclonals and polyclonals but I would suggest pAb to assure cross-reactivity with mRuby3: https://www.abbkine.com/?s=RFP-tag&post_type=product&submit=
Michael, thanks for your response. I realize that an anti-RFP antibody could cross-react with mRuby. But I was specifically looking for an antibody that someone had prior experience using and that had been used in a publication. Keagan, to answer your question, no. I haven't found one yet. I stopped looking a long time ago. I used an alternative approach for my experiment.
Hi Jyoti, thanks for your response. Fair enough, it does seem there is no specific antibody to mRuby. Do you mind telling what you did instead?
I inserted a different small epitope tag after mRuby which could be easily detected using a commercially available antibody.
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