2D PAGE problem

p.bayliss at utoronto.ca p.bayliss at utoronto.ca
Sun Jan 11 00:21:38 EST 1998

  When I run the first dimension of a 2D gel on this particular sample,
something stacks inside the tube (visible by bromophenol blue not getting
through it).  If left long enough (eg. 4hr) and with less sample, it
eventually runs out of the bottom of the tube.  It's a nuisance, and may
be taking some of my protein with it (especially LMW), and it seems to
temporarily shift the pI gradient (bromophenol blue which DOES get past
turns yellow, even if it's not near the bottom of the tube).  With silver
stain, it doesn't stain like normal protein, but stains a very light
blue.  I haven't stained with coomassie.  In the second dimension, it
again all runs to more or less the same spot.  I'm thinking it's lipid,
but I'm not sure.  I've tried acetone and also chloroform extraction with
minimal success.  I'd like to rid myself of it altogether.

  Has anyone had similar problems, or have any suggestions?  Anyone know
of a really good way to solubilize proteins and then get rid of lipids?

     Thanks in advance for your help!!

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