LOGAND <logand at msdos.ensam.inra.fr> wrote:
>Tanya Shang wrote:
>>has anybody done western blotting with those Pharmacia Phast protein
>>electrophoresis gels? The phast system separates proteins very well, but
>>the gels used on it have a plastic plate attached, can they still be
>>blotted with the conventional capillary method? I know that Pharmacia
>>sells a separate kit for western with Phast systems, but I am only doing
>>a few experiments so I don't want to buy it. If anybody knows of a
>>method to blot Phast gels without the kit and is willing to share the
>>information, i would be very grateful.
>All you have to do is remove them from the backing support. You can use the LKB
>(Pharmacia) Filmremover - essentially just a curved surface with a cheese wire
>attachment which slides between the acryl and the plastic backing - you could
>cobble together a similar apparatus using a piece of fuse wire. Once you have
>the separated acry gel blot as normal (though for less time since gel very
>David C. Logan
I agree whole-heartedly with David's suggestion. In fact, as thin as
research money is, we couldn't afford the $400 "blotting apparatus" so
I simply did like David Logan did and made a homemade job. Basically
I stripped the insulation off of some thin copper wire and looped it
around a pipet tip box lid. (I had first cut a semi-circle in the
short side of the end of the lid and then cut notches about 1/2 inch
from the same end on the bottom.) For a curved surface, I cut a "V"
in the side of an old plastic Tris bottle to "hang" the "handle" of
the Phast gel on. Having wedged the gel in place I then push the tip
box lid apparatus wire down at the interface of the stack and the
resolving gel and pull slowly down with "gentle" pressure to remove
the gel from the backing. I've done this a couple of dozen times or
so, now and I've never had any problems. We routinely use the 8-25%
gradient gels and there is always some residual acrylamide left on the
backing at the higher concentration end of the gel--this doesn't seem
to be a problem. Occassionally I break the wire by pushing too hard.
It really doesn't take much pressure. It's a quick fix to tie on a
new wire--I keep lots of spares around.
As for blotting...we work with a 10 kDa protein which migrates into
the higher concentration of acrylamide. We use a semi-dry blotter and
it never takes more than 15 min to transfer a phast gel to
Hope this helps. If you have any questions or suggestions to improve
our backwoods method, please email me!
TAMU Dept of Biochemistry and Biophysics
davesmith at bioch.tamu.edu