Message-Id: <l03010d01af028c48746c@[131.111.47.52]>
In-Reply-To: <199701151142.MAA16493@garm.adm.ku.dk>
Date: Wed, 15 Jan 1997 13:39:39 +0000
From: Len Packman <lcp2@mole.bio.cam.ac.uk>
Subject: MassSpec:MALDI from blots
To: Recipients of ABRF List <abrf@aecom.yu.edu>
>Does somebody know a method or have some suggestions, how to do
>MALDI on proteins blotted to PVDF membranes and CBB-stained.
Arne,
Dr Christoph Eckerskorn is one of the best authorities on this subject. His
email is eckerskorn@genmic.biochem.mpg.de (Christoph Eckerskorn).
>From his talks, I think you will find it is bad news to stain the blot at
all. It is best to keep it wet and locate the protein by staining a side
strip. A literature search for Eckerskorn and F. Lottspeich will produce
appropriate papers, but here's a recent one
TI: ULTRAVIOLET MATRIX-ASSISTED LASER-DESORPTION IONIZATION-MASS
SPECTROMETRY OF ELECTROBLOTTED PROTEINS
AU: SCHREINER_M, STRUPAT_K, LOTTSPEICH_F, ECKERSKORN_C
JN: ELECTROPHORESIS, 1996, Vol.17, No.5, pp.954-961
AB: Direct mass spectrometric analysis of proteins electroblotted onto
polyvinylidene fluoride membranes after sodium dodecyl sulfate-
polyacrylamide gel electrophoresis is demonstrated by matrix-assisted
laser desorption ionization-mass spectrometry (MALDI-MS) with a
linear time-of-flight instrument, equipped with a nitrogen laser (337
nm). The blotted proteins were desorbed directly from the blotting
membrane after incubation with sinapinic acid as matrix. Different
commercially available membranes resulted in high quality protein
signals for hydrophobic membranes exhibiting high specific surface
areas (Immobilon PSQ or Trans-Blot) of for charged membranes
(Immobilon CD). Systematic investigations with standard proteins were
performed to compare standard preparation procedures for ultraviolet
(UV) MALDI-MS on stainless steel sample stages and preparation of
proteins immobilized onto membranes either by direct application from
protein solutions (spotting) or by electrotransfer from gels
(electroblotting). Aspects such as mass resolution, reproducibility
from shot to shot and spot to spot, mass accuracy, and preservation
of protein localization are addressed in this paper
Good luck
Len
******************************************************
Dr Len C. Packman
Assistant Director of Research
Protein and Nucleic Acid Chemistry Facility
Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW, UK
Tel: +44 (1223) 333639 (including answerphone)
FAX: +44 (1223) 333345
e-mail: lcp2@mole.bio.cam.ac.uk
Visit my WWW page at http://www.bio.cam.ac.uk/proj/adr/PNAC/pnac.html