Elution concentration of proteins cut from two-dimensional polyacrylamide gels using Pasteur pipettes.
The present study devised a new procedure for concentrating proteins cut from primary two-dimensional polyacrylamide gels prior to their structural characterization. Gel spots containing a protein were cut from several identical two-dimensional gels and loaded on the top of a high tensile strength stacking gel in a Pasteur pipette. The protein was concentrated electrophoretically into a small volume in the narrow tip of the pipette. The concentrated protein was then blotted from the stacking gel onto a polyvinylidene difluoride membrane, where the protein was subjected to tryptic on-membrane digestion. Utilizing this system, we identified 22 proteins chosen randomly from two-dimensional gels of proteins from rat dermal papilla cells by internal microsequencing. As much as 1.5 mL volume (cut gel spots in protein sample buffer) could be loaded onto the Pasteur pipettes, generally yielding a final on-membrane area of approximately 2 mm2 after elution concentration and electroblotting onto polyvinylidene difluoride membranes. We concluded that this newly devised system is effective and useful for concentrating proteins prior to structural characterization, and that larger volumes than previously recommended can be effectively concentrated, with little or no effect on the final on-membrane area occupied by the concentrated proteins.
Kristensen
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DB
Yoshizato Morphomatrix Project, ERATO, JST, Kagamiyama, Higashihiroshima, Japan.
Inamatsu
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M
Yoshizato
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D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents; D015180:Electrophoresis, Gel, Two-Dimensional; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011145:Polyvinyls; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D051381:Rats; D012394:Rosaniline Dyes; D017421:Sequence Analysis; D012867:Skin / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism; D005465:Fluorocarbon Polymers
D004396:Coloring Agents; D008567:Membranes, Artificial; D010446:Peptide Fragments; D011145:Polyvinyls; D011506:Proteins; D012394:Rosaniline Dyes; D014357:Trypsin; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C004692:coomassie Brilliant Blue