Anti-GSK3 beta antibody (ab131356)

At Abcam we generally do not recommend re-use of the diluted antibody as signal quality may decrease. However if you were to do this, use of 0.05% sodium azide should be enough to keep any bacterial growth down. When using sodium azide in this solution please be sure to wash the blot thoroughly to remove any residual azide prior to the addition of any HRP conjugated secondary antibody as sodim azide will interfer with HRP detection systems.

There are generally a few reasons why doublets form in western blot. One is that the sample has undergone proteolysis, for this we recommend creating new samples using fresh buffers with new protease inhibitors includes and to keep the samples cold at all times during preparation. Another cause may be the existence of isoforms of the protein of interest. In review of GSK3 beta I do see that a second isoform exists which has only a 1.3kDa difference in size. As each antibody is directed against regions which are the same in both isoforms, it may be that your blot is showing the two isoforms present in your samples. We would recommend repeating the experiments using 293 cell extracts, treated with insulin (40µM, 30min) as a control alongside your samples and to use fresh buffers, protease inhibitors while keeping the samples cold.