>I would appreciate it if anyone who has experience purifying or running
>lc/ms on large fragments from CNBr or tryp cleavage would share what resins
>and buffers they have used.
>
We've done a fair bit of CNBr cleavage over the years, followed by C18
purification of the cleaved peptides. While we didn't do an exhaustive,
systematic study, we did adopt a prefered solution for redisolving the
peptides. As a general rule (always exceptions), it gives excellent
recovery of most peptides, even fairly hydrophobic ones. Around the lab it
is known as "supersolvent":
6 M Guanidine HCl
500 mM Potassium Phosphate
pH 2.5
To make it up, start with the correct volume of phosphoric acid and use KOH
to bring the pH up to the desired 2.5.
I don't pretend that there's a rational basis for the gemisch; it evolved
and seems to work for us.
We're only beginning to get into the LC/MS side, but so far we're pretty
happy with using this as the loading "buffer" for the peptides. We still
prefer the Vydac C18 columns, the 218TP series.
Good luck
Rod Levine
NIH
Bldg 3, Room 106 MSC 0320
Bethesda, MD 20892-0320
email: rlevine@nih.gov
voice: 1 (301) 496-2310
fax: 1 (301) 496-0599