HI
I have been trying to work with Er alpha MC-20 from santa cruz. It fails to detect a band. I did follow the dilution factor provided by the protocol. Can anyone suggest me if they have performed western blotting with ER alpha?
Western blot protocols usually require some optimization. You should try different concentrations of antibody, ug of initial protein loaded and maybe try different blocking solutions (milk, BSA, FBS...).
My experience with the antibodies of that commercial house is that if for example it says a dilution 1/4000 I can visualize the bands of interest with dilutions of 1/1000 or 1/500. We happened to detect IgG in sera derived from dogs against a Trypanosoma cruzi antigen. My advice is to establish your optimal test conditions, perform a pilot test, including a positive control.
The other thing to take care of is that these dilutions can generate background noise in immunodetection.
Verify dilutions of primary and secondary antibodies.
Successes.
Hi Thapa, I agree with above answers, to add to the discussion, many at times antibodies from CST and Genetex worked well for me. as advised previously, cell specific optimization is a good idea (with different concentrations). Based on my experiance, having a known positive control alongside in your blot would definately answer many of your 'if's and but's' especially in western blotting!. Good luck.
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