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Re: technical problem



I once experienced this problem when overlaying the running gel with
organic solvent,
i.e. isobutanol satuarated with buffer.  The solution: don't overlayer.


>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. 



Michael Gribskov  -  http://www.sdsc.edu/~gribskov  -  gribskov@sdsc.edu
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