Retinoic Acid Receptors Protein Extraction - (Feb/21/2007 )

I've been trying to see RAR proteins using Westerns in MCF7 cells now for a while and have had limited success with RARα, but absolutely no success seeing RARβ. I've tried loading nuclear extracts as well as whole cells and am using a Santa Cruz antibody that other publications use, but with little success. I get a lot of background, so it's either because of that that I can't see the correct band or something is going wrong with the protein extraction. What I do know is that my Westerns look nothing like published ones!

If anyone has any experinece in this field, I would love some advice!

Hi,
I worked in a retinoic acid lab for a number of years. We never got the
anti-RAR from Santa cruz to work. We used one from Pierre Chambon.
This is no help to you.
But, I think the antibody from Affinity Bioreagents works okay.
Also, do a blot with a cell line expressing high levels of the RAR
and one that has NO RAR and compare the banding patterns.
It should be clear what the RAR is. Also, induce the receptor with ATRA for a couple of hours and it should go up significantly. Another good control.
I always used nuclear extracts for my Westerns for RAR.
I don't know about ERbeta.

I've been trying to see RAR proteins using Westerns in MCF7 cells now for a while and have had limited success with RARα, but absolutely no success seeing RARβ. I've tried loading nuclear extracts as well as whole cells and am using a Santa Cruz antibody that other publications use, but with little success. I get a lot of background, so it's either because of that that I can't see the correct band or something is going wrong with the protein extraction. What I do know is that my Westerns look nothing like published ones!

If anyone has any experinece in this field, I would love some advice!

Thanks for the advice. I'm currently transfecting some U2OS cells with an RARβ plasmid and using this as a positive control. I will look into the other antibodies as well.