I suspect that you do not have 20 ug in your band, simply because most
users overestimate the amount of protein by at least 10 fold. If you stain
your gel in Commassie with 0.2% Commassie, 20% Methanol and 0.5% Acetic
Acid, and then destain with 30% methanol with no acetic acid I think you
should not get so much shrinkage that you cannot see the band. If you have
other bands so close that they overlap you will need to work on either your
gel percentage or your protein purification because you will probably have
some of the second protein within your band. Let your facility wash the
gel peice with ammonium bicarb themselves before starting the in-gel
digest.
If you are unsure about how your proceedures and your facility's
proceedures are meshing, you may want to run BSA on a gel, cut it out and
send it in for internal sequence. Also, to know how much material there
really is on a gel, run dilutions of molecular weight markers (known
amounts of course) on the same gel as your sample and estimate the quantity
of your sample by eyeball comparison. It will be low if your protein is
heavily glycosylated, but hey, more is better for an internal digestion.
If you have already determined that you have plenty of protein by amino
acid analysis of a fragment of your gel peice you may indeed need to
de-glycosylate your protein. I would try the protocols in "Current
Protocols in Protein Science" but I myself have not used any of them. My
suggestion would be to rule out the simple stuff first.
-laurey
Weather Inconsequenctial: Beautiful day after a stormy night. Now if my
kids would just get well, all would be fine!
Laurey Steinke
Protein Structure Core Facility
University of Nebraska Medical Center
Omaha Nebraska, 68198-4525
Phone (402) 559-6647
FAX (402) 559-6650
lsteinke@molbio.unmc.edu