need help-mysterious problem

Tina anfortinjp at webnet.qc.ca
Tue Dec 17 20:49:32 EST 2002


Hi everybody,

I have a mysterious problem with simple SDS-PAGE (mini gels). I load the
wells with boiled samples (cell lysates) and started migration (4 mini-gels
0.75 mm, 90 V, 2 hours). I loaded for example 8 wells of the ten available.
After this I stained the gel with Sypro Orange (and took a photo) in order
to see if I have the same loading in each well (20 ug total proteins/ well).
Each time the 2-3 central lanes are straight but the lateral lanes are
curved toward the sides (convex look). Moreover there is a very small space
between lanes, more than the space between starting wells. So, after
transfer and immunodetection with antibodies (Western Blot) the bands in the
first and last lanes are larger than those of central lanes (not suitable
for further densitometry!!). Immediately after I put the power supply ON for
the migration, the samples go out the wells toward the bottom of the gel
(that's OK) but also at left or right, like an overflow. Proteins migrated
approx. straight at the beginning, continued toward the exterior and
returned toward the interior (enlargment then shrinking).

To resolve this problem, I tried the following things:
- electrophoresis solutions of other person in the lab
- samples (cell lysates) of the same cell type from other person in the lab
- a different cell type
- a different electrophoresis apparatus (different company)
- another power supply

What's happen ??? I'd never seen this in the past !!!

Any hint will be very appreciated!
Thanks a lot in advance!
Tina





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