I'm looking at levels of both Estrogen Receptor proteins in MDA-MB231 following a treatment. ERß has been fairly easy to probe for in Westerns with a rabbit polyclonal antibody I got from thermofisher. I got clear distinct bands. Probing for ER alpha, however, has been an absolute nightmare. I've tried three antibodies so far: the F-10 monoclonal mouse from santa cruz, the SP1 rabbit monoclonal from thermofisher (249R-1) and the Rabbit polyclonal from millipore (06-935). I have gotten a lot of non-specific binding with some of them, but no band around the expected ERα size... not even in the positive control (MCF-7). I've recently tried subcellular fractionation and ran a wb using nuclear lysates but it didn't help. Nothing in the MCF-7 lane either. My secondaries are solid, and my buffers have worked for me so far, looking at other proteins both nuclear and cytosolic. Any advice would be greatly appreciated.
https://www.scbt.com/scbt/product/eralpha-antibody-f-10
Triple negative breast cancer cells (MDA-MB-231) do not express estrogen receptors. So, you may not get any band there. In case of ER positive MCF-7 cells you are supposed to get positive bands for estrogen receptors. Treatment with 17-beta estradiol may induce the level of expression estrogen receptors in MCF-7 cells. This may be an option for you.
Tapan, MDA-MB-231 DO in fact express ER alpha, just not enough to be classified as ER+. There are several publications out there with westerns with an ER alpha band in MDA-MB-231s.
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