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Daniel Kerr 03-09-2006 06:05 PM

7kDa protein electrophoresis
 
I'm trying to detect a 7kDa protein by Western Blot.

I've ran the cell lysate in a 15% acrylamide gel and the problem is,
proteins bellow 14kDa just vanish.

The Molecular standard is broad range (6-150kDa) and even the weight
doesn't show in coomassie or in the ponceau (I've stained half the gel
and transfered the other half).

Does anyone has a tip or similar problem?

Thanks


DK 03-10-2006 04:48 AM

7kDa protein electrophoresis
 
In article <[Only registered and activated users can see links. Click Here To Register...].net> , "Daniel Kerr" <[Only registered and activated users can see links. Click Here To Register...]> wrote:

Bis-Tricine gels have been developed precisely to combat a
problem of poor resolution of small proteins/peptides.

If your problem is actually a poor retention on membrane
(make sure it is not a poor transfer first!), switching to a
lower porosity (0.2 um) and higher capacity (PVDF)
membranes combined with the removal of SDS from gel
(15-30 min soak in transfer buffer before assembling
the sandwich) should help. May also try increasing MeOH
in transfer buffer to 30%.

DK

Dr Engelbert Buxbaum 03-16-2006 03:49 PM

7kDa protein electrophoresis
 
DK wrote:



And of course: leave out the SDS from the transfer buffer. SDS increases
mobilisation of large proteins, but reduces binding to the membrane.
MeOH increases binding of small proteins to the membrane, but reduces
mobility. Thus only one or the other are to be used, not both at the
same time, as commonly seen.


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