发信人: royluo (roy), 信区: Biology
标 题: Re: how to desalt from peptides mixture?
发信站: The unknown SPACE (Sat Aug 17 23:23:39 2002), 站内信件

I have similar experiences as yours. That is about determination
the anomericity of glycosylation on EGF repeats. It involves
processing the small domain EGF into peptides, and then subject them
to glucosidase digestion.
I would suggest you using HPLC with either Reverse Phase or Gel filtration
column(like superdex from Pharamacia). This is the best way to recover
small amount of peptides. If your sample has quite a lot and the MW is
big enough, you can try acetone precitation to get rid of salt. But in
your situation, it is not gonna work. Moreover, after every HPLC purification,
you will lose some protein, sometimes >50%,especially when peptides are
very small (they easily get stuck on the surface of tubes). Running HPLC
takes way too much time, but we have no choices. In my experiments,
I run almost more than 10 times for just desalting!
As to the second the question, it is probably necessary to remove
salt. At least I did this. I use urea, and NH4HCO3 as digestion buffer,
use iodoacetamide as alkylation reagents.




【 在 createrxm (coughfish) 的大作中提到: 】
: Three step reactions of peptides, every step will use new buffer and requir to
: remove all salts used in the previous step. But my sample isn't that much, so
: the lysis (SHEN XI) isn't practible. Also, try to use microcon, but the
: samllest one is 3000 cut off, still too big. HPLC seems working, but it takes
: long time, and every time have to dry the sample volume down which need extra
: several hours waiting. Any suggestions?
: Also, similar problem after reduction and alkylation of peotein, have I to
: remove the salts before trypsin digestion? I use NH4HCO3 as digestion buffer.
: Thanks a lot!


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