multiple sequences from Edman degrad
xyzzyx at aimnet.com
Fri Oct 27 01:42:18 EST 1995
In article <24OCT95.19774843.0038.MUSIC at SEMOVM>, C478SCB at SEMOVM.SEMO.EDU
>I had a brain spasm regarding generating multiple sets of sequence
>data from a series of manual Edman degradations, and wanted to see
>if anyone would be good enough to tell me why this can't work.
>I want to purify some intracellular fungal proteases and determine
>enough sequence data to design degenerate probes. I have the purif.
>worked out and am going to do the sequencing manually. It seems to
>me that I should be able to determine 10-12 N-termnal residues before
>the background gets to be a problem. Since the activities that I am
>interested in are a royal pain to isolate, I'd like to cleave the
>bulk of the remaining polypeptide with CNBr, purify the fragments
>and get some internal sequence data from one or more of these pieces.
>Think it will work? Any comments wil be greatly appreciated, especially
>as I've never done any sequencing and can't afford to send the samples
>off for out of house sequencing.
>c478scb at semovm.semo.edu
whether or not you are successful depends on a lot of things. I ran
a protein sequencing lab for a few years, so I guess I know a FEW things about
this. Manual sequencing is a heck of a lot less efficient than automated,
gas-phase sequencing (e.g. the ABI 477A or newer machines). We routinely got
95+% yield/residue (except for the weird ones). We routinely ran out to 30
residues, and could usually successfully call most of them. The problem is
that the "background" picks up, as the residue signal diminishes. Eventually,
between noise, previewing, postviewing, termination, etc. there was no
discernable signal. BTW, we usually started with ca. 100 pmol protein. I'd
guess that manual sequencing will give you 85-90% repetitive yield (or less)
at best, so your odds of pulling this off depend very much on how much sample
you have to start with. The same holds true for your CNBr peptides. Make
sure you completely desalt everything, before you start and use really good
TFA and PITC (you'll regret it otherwise), Pierce is a good, reliable source.
I'm sorry that I no longer run that lab, so I can't help you out here.
I urge you to call in any favors that you can to get an automated sequence
run, if at all possible.
One person you may want to give a call to is Dr. Steven Clark at (I
think it is now called Syphergen, they used to be called Abiotic Systems).
Their number is 415.496.3771. I put in a VERY brief stint as a consultant
for them. Steven is working on a really nice Edman sequencing system that
utilizes MALDI-TOF MS. The last time we spoke, he was working down in the
femtomole (!) range. He might be able to help you out, he seemed interestd in
new and unique proteins.
Matrix Pharmaceutical, Inc.
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