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SDS PAGE - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 11-30-2006, 09:02 PM
younes sadramehr
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Hi ..........
We had run a total protein SDS PAGE . After staining we saw a new & different band in one of the PAGE columns . Now we want to repurificate the protein ( the new band ) from the poly acrylamide gel . how should we do it ??we need the protein to re run on low voltage SDS PAGE for better isolation . how can we elute the protein in an economical & fast way ???/
thanks for your help .



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  #2  
Old 12-05-2006, 10:37 AM
Dr Engelbert Buxbaum
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younes sadramehr wrote:


Cut out the stained band, mince the gel and pack the slurry in an
electroelution tube. The protein and stain will move from the gel and
collect above the semipermeable membrane at the bottom of the tube. From
there they can be withdrawn with a fine needle.

If you don't have elecroelution facilities you can press the gel slice
through a 27G needle and incubate the slurry with buffer solution in an
end-over-end mixer overnight. 10 mM Bicarbonate pH 8 or similar should
do. Spin the gel fragments down and withdraw the supernatant. Recovery
will be lower than by electroelution.

Another idea would be to do a western blot (possible with stained gels)
and cut the band from the membrane (PVDF, not NC!). N-terminal gas-phase
sequencing of the protein can be done directly with those membrane
slices, identification of the protein from the partial sequence is then
done by a data base search. There are commercial sequencing services
available which are quite affordable.
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  #3  
Old 12-05-2006, 07:21 PM
Tom Anderson
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On Tue, 5 Dec 2006, Dr Engelbert Buxbaum wrote:


A colleague of a colleague used to do this by cutting the end off a 1000
ul filter tip, putting it in a microtube, putting the gel piece on top of
the filter, and spinning it. Apparently this works, although i dread to
think what the yield is like.

tom

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