Dye-free protein molecular weight markers.
Protein molecular weight markers are widely used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Here, we describe novel protein molecular weight markers in which a prestaining procedure is no longer needed. Green fluorescent protein (GFP) is stable and resistant to denaturing agents/conditions. Various histidine-tagged GFP fusion proteins were overexpressed in Escherichia coli and purified by metal affinity chromatography. The minimal amount of each protein marker needed for analysis in SDS-PAGE and Western blot under visible light was 62.5 and 125 ng, respectively. Under ultraviolet (UV) ray, the minimal amount of each protein marker needed for analysis in SDS-PAGE and Western blot was half of those amounts used under visible light, respectively. Collectively, the accuracy, sensitivity, ease, economy, and flexibility of our strategy may reinforce the application of GFP in molecular biology.
Chang
Microsugar
M
Institute of Biotechnology and Department of Life Science, National Dong Hwa University, Hualien 97401, Taiwan.
Hsu
Hsue-Yin
HY
Lee
Han-Jung
HJ
D015153:Blotting, Western / Q000379:methods; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase