Two-dimensional polyacrylamide gel electrophoresis of proteins labeled with the fluorophore monobromobimane prior to first-dimensional isoelectric focusing: imaging of the fluorescent protein spot patterns using a cooled charge-coupled device.
A new method for visualizing 2D protein spot patterns is described whereby proteins containing sulfhydryl groups are labeled with the fluorophore monobromobimane prior to the isoelectric focusing step of 2D polyacrylamide gel electrophoresis. The method requires the addition of a single reagent and a delay of only 15 min during sample preparation. High resolution spot patterns were obtained without the need for any post-electrophoretic gel handling. The gel patterns were imaged digitally immediately post-electrophoresis while the gels were still within their glass electrophoresis cassettes. The imaging system was based on a cooled charge-coupled device. The fluorescent spot patterns of proteins from the lymphoid cell-line IM9 were similar but not identical to silver-stained gel spot patterns of the same but underivatized sample. There was an overall increase (approx. 10%) in the number of spots visible on the fluorescent gels compared to those visible on the silver-stained gels.
Urwin
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VE
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, United Kingdom.
Jackson
P
P
D001643:Bridged Bicyclo Compounds; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007525:Isoelectric Focusing; D008221:Lymphoid Tissue / Q000737:chemistry / Q000166:cytology; D011506:Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis; D016622:Silver Staining; D013438:Sulfhydryl Compounds / Q000032:analysis
D001643:Bridged Bicyclo Compounds; D005456:Fluorescent Dyes; D011506:Proteins; D013438:Sulfhydryl Compounds; D012710:Serum Albumin, Bovine; C028381:monobromobimane