I have been trying to follow a R. Burgess paper in which enzymatic
activity was regained in a protein purification after crushing the
suspected protein band from the gel. The problem that I have been having
is the transient staing of the gel to identify the approx Mr prior to
cutting out the band. The original paper recommends ppt the SDS in the
gel with 0.2M KCl for 5 min and then destaining with water for up to one
hr. When I tried this the portion of the gel below the dye front became
very white, implying ppt, but the portion of the gel above the dye front
only became slight opaque and I could only see very faint bands of heavly
overloaded lanes. I was made aware of another transient staining tech
using a 0.3M copper soln which I tried on the same gel with poor results
(although this gel would not fully destaing from the KCl treatment and
that probably interfered).
I would appreciate any advice/procedures that you may have successfully
used or heard about. Thanks in advance!
