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technical problem



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.