Monday morning I came into the lab and began developing last weeks membranes for western analysis. I used DAX1 as my primary antibody, since the gel contained a lane of isolated DAX1. Unfortunately, the IEF gel markers were the only bands to appear. At this point, neither Dr. Clendening or I had any idea as to why the transfers were unsuccessful.
Phone Call
Dr. Clendening and I decided it would be best to call up the company that provided us with our IEF gel and try to troubleshoot. The woman that I spoke with recommended using the acetic acid transfer, but letting it go overnight. She said that she would send us a gel right away.
IEF Again
When the IEF gel came in, we decided to run last week's experiment again. Samples of the nuclear fraction, nuclear fraction with formaldehyde/glycine treatment, and isolated DAX1 were loaded into the gel. Experimental conditions were kept constant, except we ran the transfers overnight. I developed the westerns the following day, but we got the same result as before. DAX1 was not detected in any of the samples.
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