I like lambda phosphatase from calbiochem. 1 ul of peptide/tryptic digest,
a little bit of tris to get pH to ~ 7.5-8 (hepes works too) low mM ~ 5 or
so. enough MnCl2 for ~ 1mm and about .1 ul lambda phosphatase. 15 minutes
rt. Phosphates are removed (from peptides). It's not so easy for proteins
though. I examine peptides using MALDI-TOF so if you are using ES some of
the salt, glycerol and other things that the enzyme is in may cause
interference.
-----Original Message-----
From: Nicola O'Reilly [mailto:oreillyn@icrf.icnet.uk]
Sent: Wednesday, February 09, 2000 7:30 AM
To: Recipients of ABRF List
Subject: Removing phosphate from peptide
Dear All,
Has anybody used alkaline phosphatase to remove a phosphate group from a
peptide? I would like to remove phosphate from a serine or threonine
residue then mass analyse. I would like advice on
a) where should I get my alkaline phophatase from & which formulation I
should use (Sigma have a bewildering variety of alkaline phosphatase
preparations).
b) How many units of enzyme per amount of peptide, buffers, times, temps
etc.
Thanks for your help with this
Nicola O'Reilly
Weather Report.
A weak sun is shining in London & the crocus are blooming in Lincoln's Inn
Fields.
Nicola O'Reilly
Peptide Synthesis Laboratory & 2-D Gel Electrophoresis Laboratory,
Imperial Cancer Research Fund.
44 Lincoln's Inn Fields, London, WC2A 3PX.
Tel 0171 269 3318 or 0171 269 2886
Fax 0171 269 3579
This archive was generated by hypermail 2b29 : Mon Feb 14 2000 - 16:05:59 EST