Hi there, back again with another question about SDS-Page / Western blotting.

I am planning to load 30 ug of protein onto a 5-20% gradient gel. Protein of interest (multiple) 260kd, 62kd, 40kd.

Run the SDS-page at 10mA until the separating gel, turn it up to 30/40 mA.

Membrane activation for 1min with methanol, wash with water before stacking on the sandwich.

Use Tris -glycine buffer (20% methanol). (no sds).

Since my protein of interest is quite big I'll transfer overnight at 30V Constant voltage.

after transfer there seems to be a few lanes of protein still in the gell.

The gel included in the picture was ran at 100V for 1hr. (homemade gel and different protein of interest). But the same has occurred for the gradient gel with slight bands being left behind in the gel after overnight transfer at 30V CV. Can somebody please give me some hints or tips. (PS i found out that my sample prep wasnt good and that could have caused aggregation, but this shouldnt affect transfer right?)