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To prepare my sample, I used a buffer containing 50mM Tris pH 7.5, 150mM NaCl, 1mM MgCl2 and 1% triton X-100. I included proteases inhibitors in the lysis buffer (complete edta-free tablet, from Roche). I incubated the cells 20 minutes with
this buffer and then cleared the lysate by a 15 min centrifugation at 20 000g. I evaluated the protein concentration of my
samples by the bradford method and then I added SDS-sample loading buffer to the lysates (containing DTT). I boiled the
samples for 5 minutes.
I suppose that the 8% acrylamide gel I used for SDS-PAGE is suitable for the experiment I performed since I see the 31kDa
band of the marker, meaning that proteins around 48 kDA might still be in the gel, although I could also try with a 12%
There was no SDS in my transfer buffer, only methanol, tris and glycine. I did a Ponceau coloration of the membrane to
make sure the transfer worked well and everything was normal. I can also precise that I used these samples for other gels
that were runned and transferred at the same time as the gel described for the western blot that did not worked with
ab12875. I was able to detect 3 other proteins by Western blot, the only experiment that did not work was the one using
ab12875 primary antibody. Those proteins had a size of 60, 55 and 42 kDa so there is no reason why I should not see Aurora A which is supposed to migrate at 48 kDa.
We purchased this antibody less than one month ago so it might be possible that we request a credit. According to the catalog, Abcam sells other anti Aurora A antibodies that were reported to work well in western blot experiments so we would
be interested in trying another one.
Asked on Oct 18 2012