Concentration of, and SDS removal from proteins isolated from multiple two-dimensional electrophoresis gels.
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.
Dainese Hatt
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Protein Chemistry Laboratory, ETH-Zentrum, Zürich, Switzerland.
Quadroni
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Staudenmann
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James
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P
D000595:Amino Acid Sequence; D018833:Chaperonins / Q000737:chemistry* / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional; D005960:Glucosides / Q000737:chemistry; D008969:Molecular Sequence Data; D012231:Rhizobium / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000032:analysis* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005960:Glucosides; C018619:octyl-beta-D-glucoside; D012967:Sodium Dodecyl Sulfate; D018833:Chaperonins