At 06:41 PM 28-04-2001 EDT, User2105494@aol.com wrote:
>1. All of those stories that you heard about how acrylamide was a bad thing
>to expose a protein to were wrong. Actually, a lot of the historical "don't
>do" warnings about electrophoresis as related to Edman degradation were
wrong
>and got into the dogma anyway, but since no one is interested in Edman
>degradation anymore, I guess we don't need to worry about it either.
>
>2. The reaction of acrylamide with the N-terminus is very slow as compared
to
>the reaction with Cys, where slow means that you would need to leave the
>protein at room temperature in 1 M acrylamide for days before a
>significant/substantial reaction with the N-terminus was observed.
>
>3. When acrylamide modifies the N-terminus, you get preview, not N-terminal
>blockage. When the N-terminus is modified by acrylamide and the protein is
>subjected to Edman degradation, the modified N-terminal residue is
eliminated
>during the initial PITC coupling steps and the next residue is then
>derivatized. The same is true for 4-VP and 2-VP.
>
>4. Acrylamide modification of Cys in commercial gels is very common.
>Regarding the extent of modification of your protein, it usually has a lot
to
>do with how much protein ran ahead of your protein. The first protein to
>"sweep" the lane of acrylamide usually has a high percentage of the Cys
>residues modified and those following it have lower and lower levels of
>modification. It's just a stoichiometry thing, since there is only so much
>free acrylamide in the lane. In the olde days, people added thioglycolic
>acid to the sample buffer to sweep the lane of acrylamide.
>
>5. As pointed out by Ken Mitchelhill in the ABRF discussions a while back,
>acrylamide is an excellent pre-electrophoresis Cys alkylating agent. His
>protocol can be found in the electronic archives.
>
>6. (I lied, there's a 6th point) Acrylamide may be the best Cys alkylating
>agent on the market. The reaction goes quickly and in good yield.
>Acrylamide is also thought to be able to access Cys residues in more
>restricted or hydrophobic environments than other alkylating agents. Also,
>acrylamide doesn't stink, but if you're using TMA, the VP's probably don't
>bother you. Dimethylacrylamide is also a good alkylating agent and is a
>liquid at room temperature (easy to pipet). The dimethylacrylamide adduct
of
>Cys elutes much later in the chromatogram.
>
>Hope that helps,
>
>Steve Tindall
>Argo BioAnalytica Inc.
Many thanks to all that replied to my query...I've learned a lot. If it's
not free acrylamide blocking the N-terminals, does anyone know what is
doing the deed? Presumably it's still a good idea to prerun gels for low
level samples.
I'm really sorry to hear that nobody is interested in Edman sequencing any
more though...I'm obsolete!
Regards,
Sandy
Sandy Kielland
Victoria Protein Microchemistry Centre
Department of Biochemistry and Microbiology
University of Victoria
Victoria B.C. CANADA
phone: (250) 7218884
FAX (250) 7218855
Email Kielland@UVic.ca
Webpage http://www.proteincentre.com
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