Running Retic lysate on SDS-PAGE

[John A. Newitt]

In article <boehnin1-210395144923 at jah1.oac.tju.edu>,
boehnin1 at jeflin.tju.edu (Darren Boehning) wrote:

> We had the same problem for some time, and solved it by incubating at 45C
> for 15 minutes after it is quenched in SDS buffer (as opposed to boiling
> the sample before running it out on the gel).  Reason for success with this
> method may be that some proteins like to aggregate at boiling temperatures
> after quenching, and therefore this is eliminated by the lower incubation
> temperature.  

I use 65 degrees for 30 minutes to fully denature proteins in SDS-sample
buffer prior to SDS-PAGE.  This treatment also reduces degradation of some
proteins that are labile to boiling (e.g. some Asp-Pro peptide bonds are
broken at pH 6.5 with boiling).  For the reticulocyte lysates, I would
also make sure that you're not overloading the gel with protein--there is
a whole lot of protein in reticulocyte lysates.

John A. Newitt  <newitt at ncifcrf.gov>
National Institutes of Health
Bethesda, Maryland  USA