Concentration of, and SDS removal from proteins isolated from multiple two-dimensional electrophoresis gels.

Détails

ID Serval
serval:BIB_59A99542D751
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Concentration of, and SDS removal from proteins isolated from multiple two-dimensional electrophoresis gels.
Périodique
European journal of biochemistry / FEBS
Auteur⸱e⸱s
Dainese Hatt P., Quadroni M., Staudenmann W., James P.
ISSN
0014-2956
Statut éditorial
Publié
Date de publication
06/1997
Peer-reviewed
Oui
Volume
246
Numéro
2
Pages
336-343
Langue
anglais
Résumé
We have developed a gel electrophoresis system that can concentrate proteins from spots cut out of up to 50 two-dimensional electrophoresis gels. During protein concentration, SDS is substituted with a non-ionic detergent (octyl beta-glucopyranoside) which allows digestion and MS analysis of the protein directly extracted from the gel without fixation or staining. The system avoids the problems associated with the digestion of dilute protein in multiple bands by (a) greatly reducing the gel volume for digestion and thus the amount of protease required, hence lowering contamination by autodigestion products, (b) reducing the volume of solvent required for extraction of protein from the gel, thus minimising loss of material to container surfaces, and (c) removing SDS which interferes with subsequent MS or HPLC analysis. The efficiency of protein recovery ranges between an average of 80% for proteins from silver stained two-dimensional gels to 90% for fluorescence and Coomassie-blue-stained gels. The method is compatible with MS analysis of very low amounts of protein from any staining system, but appears not to be useful for Edman sequencing of silver-stained or fluorescent-stained proteins since the amount of N-terminal blockage appears to increase as the amount of protein isolated from the two-dimensional gel decreases.
Mots-clé
Amino Acid Sequence, Chaperonins, Electrophoresis, Gel, Two-Dimensional, Glucosides, Molecular Sequence Data, Rhizobium, Sodium Dodecyl Sulfate, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Pubmed
Création de la notice
24/01/2008 15:46
Dernière modification de la notice
20/08/2019 14:13
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