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Re: technical problem



This problem might be due to overloading the lanes. More is not always
better.

Rudolf Meili
        Department of Biology
        Center for Molecular Genetics, Rm.228
        University of California, San Diego
        9500 Gilman Drive
        La Jolla, CA 92093-0634
        U.S.A.
        fax:    +1 (619) 534-7073
        phone:  +1 (619) 534-4825
-----------------------------------------------------

On Tue, 2 Mar 1999 sternemarr@siena.edu wrote:

> 	For many years I have used 0.1% SDS in water (or 0.1% SDS in 
> running gel buffer) instead of n-butanol.  Hope that works for you.
> 
> Rachel Sterne-Marr
> 
> On Mon, 1 Mar 1999, Grant wrote:
> 
> > Can anyone help...?
> > 
> > 
> > 	Our institute is having problems running SDS page acrylamide gels. We have
> > found that we are losing proteins in the gels (as seen when using
> > prestained markers). When the gels are Coomasie stained it shows smeary
> > lanes with no defined bands. If the gels are transfered then there is a lot
> > of protein left in the gel. It seems to be the higher molecular weight
> > proteins which are most highly affected. 
> > 
> > 	We have changed the Water, SDS solution, Tris/Bicine stock solutions,
> > Acrylamide/Bisacrylamide solution and powder, APS and Temed. None of the
> > results show any consistency therefore we have not been able to narrow down
> > the problem(s). We have also used plastic wear instead of our own cleaned
> > glasswear as we thought that it maybe some residue retained on the
> > glasswear from cleaing that maybe affecting the gels. This has not been
> > reproducibly shown to make any difference. We also have experienced
> > variablility in wet and dry transfer but we think that this seems to be
> > down to problems with the gel and NOT with the transfer itself although
> > this maybe wrong. If you could provide any suggestions then we would be
> > very grateful as we don't know what else it could be. We use a continuous
> > gel system (Hoefer SE400 electrophoresis unit and transfer tank). The
> > recipe for the gel mixture and transfer buffer are given below:
> > 
> > 6% acrylamide gel:
> > 
> > 27.4mls H2O
> > 4mls 1M Tris/1M Bicine Solution
> > 8mls 30% Acylamide solution (37.5:1 Acrylamide:Bisacrylamide)
> > 400ul 10% SDS
> > 80ul TEMED
> > 200ul Fresh 10%APS
> > 
> > Run at 30-40mA for 5-6hrs (NOT cooled)
> > 
> > Wet Electrotransfer Buffer:
> > 
> > 7litres
> > 
> > 203g Glycine
> > 40.6g Tris
> > 20% Methanol.
> > 
> > Run at 200-250mA overnight (not cooled)
> > 
> > 
> > We have been using this system for along while and only now has this
> > problem arisen.
> > 
> > Yours sincerely,
> > 
> > G.Stewart
> > 
> > CRC Institue for Cancer Studies,
> > Birmingham University.
> > Birmingham. 
> > 
> 
>