I have a transgenic mouse with a tdTomato-tagged protein, so now I would like to immunoprecipitate the tdTomato and everything that is in the complex. Three antibodies (anti-DSRed) I tested result in an awful lot of background on western blots and also on tissue sections. Does anyone know of a good antibody? Thanks, Tineke
I've tried a few, and strongly recommend the anti-RFP from Rockland Immunochemicals (ref: 600-401-379, rabbit) for immunostaining on tissue sections. Works great to detect both mCherry and Tdtomato at 1:2000 to 1:500 depending on experiment. Indeed enhances axons by eliminating the brightness difference of somas versus axons and dendrites that you normally have when looking at endogenous tomato fluorescence.
http://www.rockland-inc.com/Product.aspx?id=34801.
Hope it helps,
Dear Christina
SICGEN-Antibodies has a good one http://www.sicgen.pt/product/tdtomato-polyclonal-antibody_1_135. They also have anti-mCherry that recognizes tdTomato protein (tested by John Hopkins University).
Jose
I've tried a few, and strongly recommend the anti-RFP from Rockland Immunochemicals (ref: 600-401-379, rabbit) for immunostaining on tissue sections. Works great to detect both mCherry and Tdtomato at 1:2000 to 1:500 depending on experiment. Indeed enhances axons by eliminating the brightness difference of somas versus axons and dendrites that you normally have when looking at endogenous tomato fluorescence.
http://www.rockland-inc.com/Product.aspx?id=34801.
Hope it helps,
Thanks a lot to all. The ClonTach Ab I have, but it gives me a lot of background, especially on Western blot. But I will ook into the paper and I will look at the other suggestions, too.
Best wishes,
Tineke
RFP-Trap (chromotek); great in IP, little background! (does not work in Western)
http://www.chromotek.com/products/nano-traps/rfp-trapr/
Julien is right...the Rockland anti-RFP antibody is the best one we have tried for detecting tdTomato. We made several transgenic mouse lines with tdTomato and used this antibody for all our characterization work. The main issue is the photobleaching of native tdTomato, so it is much better to amplify the signal with the anti-RFP primary and use a more stable fluorecence-conjugated secondary antibody. I often liked to use alexa-488 conjugated secondary to change the native red fluorescence to a green signal. This gave me better resolution of fine structures like axonal projections.
Dear Christina, did you have any success in testing proposed antibodies?
Namely, I will use tdtomato for the same purpose as you and I don't know which antibodies to order..
Does anyone happen to know if this Rockland anti-RFP antibody cross-reacts with YFP or GFP?
The Rockland RFP antibody works well in tissue sections and does not cross react with GFP.
I have tried and had success with the Rockland Ab, and also with the mCherry monoclonals from DSHB@UIowa. Both recognize the tdTomato and not GFP in dot blot preps and ihc
Both the Rockland anti-rfp (Rabbit) and the sicgen anti-tdtomato (Goat) worked perfectly in PFA fixed mouse brain slices. I have not tested for cross-reaction with GFP.
Has anyone tried the Rockland Ab and know if it cross react with YFP or BFP?
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