[Date Prev][Date Next][Thread Prev][Thread Next][Date Index][Thread Index]

Re: technical problem



	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. 
>