Helene
Thioglycolic acid and 2-mercaptoacetic acid are the same chemical
There are a number of things that can cause the N-termminus of a protein to
become derivatised in the gel such that the protein can then no longer be
sequenced from the N-terminus by Edman degradation. In 2D gels, if the
first dimension is performed in the presence of urea, then alkaline
conditions can lead to the production of cyanate (from breakdown of the
urea) and this is very reactive towards primary amines, so the N-terminus
and some of the lysines will become derivatised (blocked). Also acrylamide
itself is a reactive molecule, towards thiols in particular, but also
primary amines. If a gel is not fully polymerised then free acrylamide
monomer is present which can react with the protein. Because of the higher
reactivity of acrylamide towards thiols, incorporation of thioglycolate
(mercaptoacetic) in the well buffer provides a scavenger for free
acrylamide as the thiol is mobile and moves ahead of the protein, clearing
the way so to speak, once the current is turned on. It also mops up free
oxidative radicals in the gel which can catalyse derivatisation processes.
However, these measures are most effective when the reagents used are of
high purity, the acrylamide monomer solutions are fresh and deionised with
mixed bed resin to remove acidic and basic breakdown products.
I would draw your attention to an ABRF News article on 2D gels in the March
98 issue. You can access it from http://www.abrf.org
Len
*********************************************************************
Dr Len C. Packman
Assistant Director of Research
Protein and Nucleic Acid Chemistry Facility
Department of Biochemistry
University of Cambridge
80 Tennis Court Road
Old Addenbrookes Site
Cambridge, CB2 1GA, UK
Tel: +44 (1223) 333639 (including answerphone)
FAX: +44 (1223) 766002
e-mail: lcp2@mole.bio.cam.ac.uk
Visit my WWW page at http://www.bio.cam.ac.uk/proj/adr/PNAC/pnac.html