Fluorescent monitoring of proteins during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting.
Fluorescent labeling of proteins was found to be a very sensitive and reliable alternative to conventional methods for monitoring proteins on Western blots. Proteins were labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) before SDS-PAGE. After electrophoresis and subsequent electro-blotting the fluorescent-labeled proteins were visible upon ultraviolet illumination of the nitrocellulose membranes, and could be photographed to yield an accurate record of the blots before subsequent serological analysis. The sensitivity for detecting MDPF-labeled proteins on nitrocellulose was 100-200 ng, 50 to 100 fold less sensitive than on gels. Fluorescent-labeled TMV and MStpV capsid proteins that were blotted onto nitrocellulose still reacted in serological tests and were detected when present in quantities as low as 100 pg. Fluorescent labeling allows accurate photographic records of the SDS-gel, blot and probed blot using only one sample, and no subsequent staining steps are required.
D000818:Animals; D002244:Carbon; D004396:Coloring Agents; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D005455:Fluorescent Antibody Technique; D010942:Plant Viruses / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate; D014027:Tobacco Mosaic Virus / Q000032:analysis; D014759:Viral Envelope Proteins / Q000032:analysis; D014764:Viral Proteins / Q000032:analysis
D004396:Coloring Agents; D011506:Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C028433:chinese ink; D012967:Sodium Dodecyl Sulfate; D002244:Carbon