"wrong" molecular weights - problems with sizes (Oct/13/2009 )
Why would we consistently be getting a western band that is almost twice what is expected? Essentially we are trying to do an immunoprecipitation, but are not even sure that our antibody used for the western is reactiing with the correct protein! We do our immunoprecipitation, get the proteins,and then run the western with the additional antibody. Bands are distinct, background good, etc., BUT with total cells lysates used as a control, the band seen (very intense, some additional ones, but faint) is about twice what the manufacturer says is expected. We have increased the time for denaturing-no difference. Run the exact same cells the manufactuere used, done harsh versus gently extract, no difference. WHen the total cells extracts are run with actin as the antibody, the blot is great-sizes correct, single bands, virtually no background. My next step is to get another antibody, but before I spend the money, does anybody have any suggestions?
I didn't completely get at what stage you got double bands, but still: sometimes double band can be a result of glycosilation. You can check if band size is sensitive to glycosidase treatment.