HELP: peptide separation on SDS-PAGE

L. Lintott llintott at shaw.ca
Tue Nov 27 23:38:35 EST 2001


"Krzysztof W. Wasowicz" wrote:
> 
> Hi,
> I would appreciate any help on the following topic:
> I am trying to run several neuropeptides on SDS-PAGE: neuropeptide Y
> (Peninsula), vasoactive intestinal polypeptide (Peninsula), substance
> P (Sigma) and Met-enkephalin (Sigma). These are not tissue extracts
> but just pure peptides.
> I tried different recipes for gels, and now I used method described by
> Schaegger and von Jagow (Anal Biochem, 166:368-379, 1987) - 16.5%T3%C
> separating, 10T3C spacer and 4T3C stacking gels. Schaegger and von
> Jagow claim it has good resolution from 1kDa to 70kDa. After
> electrophoresis I stain gel in regular CBB R-450 (with methanol). Now
> strange thing comes: I cannot see a band of Met-enkephalin (maybe
> nothing strange - just 0.6 kDa), I cannot see band of substance P
> (just ca. 1.3 kDa) !!!, AND  bands of neuropeptide Y (4.25kDa) and
> vasoactive intestinal polypeptide (3.3 kDa) run at EXACTLY the same
> position.
> Does anyone have some experience with separation of thus small
> peptides?
> ANY help and suggestion will be appreciated.
> Krzysztof W. Wasowicz
> Department of Animal Anatomy
> University of Warmia & Mazury
> ul. Oczapowskiego 13 (Bldg. 105J)
> 10-957 Olsztyn-Kortowo
> Poland
> Phone: +48-89-5233688
> Fax: +48-89-5234986
> e-mail: wasowicz at moskit.uwm.edu.pl
> ICQ: 58434323

I have used the trycine gel system to resolve in the 3 kDa range.  The
marker I use has 2.5 and a 3.5 kDa proteins which separate well.  I
usually run mini-gels at 100 volts for 3 - 4 hrs.  I know from
experiance that it is very important that the cathode butffer not leak
through to the anode buffer.  With older BioRad mini's this can be a
problem as they tend to leak from the upper chamber.  I always put lots
of grease on the gasket to prevent this.

I also use a polypetide fixing solution on the gel before I stain
coomassie (40% methanol and 10% acetic acid).  Recently I have
switched to using Invitrogen's "Simply Blue" and find that it works very
well for the smaller proteins, and is somewhat simpler to use. (no
affiliation).

I have no experiance myself with proteins smaller than 2 kDa, but others
tell me that the trycine gradient gels (10 - 20 %) are the way to go. 
Some company's will send you a free sample pre-poured gradient gel to
try out.

Just some thoughts,
LL




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