Hi, I've been having some problems with recombinant insulin-degrading enzyme, I have tried human and rat forms in a western blot and either nothing comes up at all, or the bands come up different on the duplicates and sometimes there is more than one band. I was thinking the diluted enzyme may not be homogenous, I am diluting in water and vortexing thoroughly before pipetting however.
The last one I did, I ran 7 serial dilutions of 200ng/well, down to 3.125ng/well, and only the 200ng band came up on 3 and 5 minute exposures, and I could just see the 100 and 50ng bands (which looked the same) on an overnight exposure. I thought that 5-50ng of purified protein was the standard range for western blots.
I have tried three different lots of the human IDE, and one of rat IDE, with inconsistent results for all. My internal standard always looks fine. Can anyone help me?