HELP: peptide separation on SDS-PAGE

Peeter Toomik peeter.toomik at ut.ee
Fri Nov 23 05:33:46 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) !!!,

Many peptides and even small proteins are soluble in acetic acid -
methanol mixture and are washed off during staining. I do not now anything
about solubility of these particular peptides, but precipitation with 10 -
20% trichloroacetic acid may help.

Peeter Toomik

> 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

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