I have been trying to optimize this new antibody I bought recently. The band size is 146 kDa. I have loaded 30 microgram of protein used a dilution of 1:500 of primary antibody. But I keep getting these unusual patterns and do not know how to troubleshoot . Any suggestions?
U-shaped bands are most commonly caused by sample overload or overheating during electrophoresis. 30ug total protein seems like a reasonable load. So try decreasing your voltage and/or cooling the slab during electrophoresis. Less commonly, high salt or lipid content can smear individual bands.
In any case, it looks like your problem probably is in the electrophoresis step, not in transfer, detection, probing, or imaging. If you want to confirm this, you might stain a gel for total protein, to obtain the same unusual band shape. If you stain with Coomassie you can still blot the gel and perform your Western.
1) Heat with 50mM DTT reducing agent at 60C for 10 min.
2) Run at 100V for 1 hour.
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