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Thank you for your kind replacement of the antibody! I was able to test it on human prostate cancer cell line PC3 lysed with RIPA buffer (last lane of the samples indicated by arrow) and my other prostate cancer cell lines prepared before. The membrane was first detected with your new antibody and then reprobed with FASN. As you can see from the images, your new antibody can only show a low molecular weight background band. FASN antibody clearly detects the target protein with several non-specific bands. The pictures in two different pages represents different exposure time. Therefore, I am afraid that your new antibody does not work either. If you can provide a positive control, I am willing to repeat experiment again. If your positive control works, it means that your antibodies work. I am happy to keep them and improve my sample preparation.
Asked on Jan 08 2013
Yes, I can send you as a one time exception a positive control lysate (rat liver (ab29413), https://www.abcam.com/liver-rat-whole-cell-lysate-normal-tissue-ab29413.html). This has been tested with ab66217 and gave strong bands.
Also I would suggest to check the gel after transfer as some of the high MW proteins might have not completely transfered onto the membrane. You can check this by staining the gel with Coomassie Blue. if you see bands in the gel at 200 kDa and higher, you might want to adjust your transfer conditions so that all high MW protein are being transfered.
Also, avoid boiling of your samples before loading them onto the gel. Warming your sample to 50-60 degree Celsius is sufficient for denaturation in loading buffer.
Again, FASN is a cytoplasmic protein and such is much easier to work with than ABCA1 which is very hydrophobic, glycosylated and of high MW.
This article and link might be of help as well: http://www.sciencedirect.com/science/article/pii/S0003269708006829
Answered on Jan 08 2013