Am 22.10.2007, 12:17 Uhr, schrieb Isabelle Holdridge <holdri39 from uwosh.edu>:
> I have been running 2D gels for soy bean total soluble proteins and I am
> consistently losing the higher molecular weight proteins. These same
> proteins are visible on the 1D gels. I have tried a variety of changes
> in my protocol, such as, changes with the running buffers, IPG buffers
> and detergents, and ph gradients, but all to no avail. The lower
> molecular weights on the 2D gel are visible and appear to be nicely
> focused. I am not getting much background staining or streaking. Any
> suggestion would be appreciated.
With so little info it is difficult to guess. What is the MW of the
proteins you are looking for? With acrylamide down to 5% 200 kDa is about
the maximum, beyond that you need to strengthen your gels with agar as
they get to soft.
How are you doing your first dimension run? I have never been able to get
ampholine tube gels to work properly, too much protein precipitates near
the starting zone. However, IPG gels work fine even with large membrane
proteins like Mdr1 (160 kDa). I use the sample solution to rehydrate the
gel (8 h at 50 V, 18 deg C), giving me a reasonable sample volume, I also
found you need about 25 kV*h to focuss a 7 cm gel, about twice of what the
manual said.
Note also that proteins with extreme pI are lost irrespective of MW.
Before the 2nd dimension run, the IEF gel must be equilibrated with sample
buffer (10 min at 37 deg C in a sealed tube on an end-over-end mixer).
This allows the detergent (SDS in most cases) to bind to the proteins in
the gel. Then mount the gel with stacking gel buffer containing 0.5%
agarose.
