研究目的
To develop a simple, sensitive, less time consuming, and economical fluorescent dye as an alternative for staining proteins on polyacrylamide gels.
研究成果
F-OB is a safe, environmentally friendly, and cost-effective fluorescent dye for protein staining on polyacrylamide gels. It offers high sensitivity, rapid staining, and reversible binding, making it a viable alternative to traditional staining methods like Coomassie Brilliant Blue and silver staining.
研究不足
The presence of SDS interferes with the binding of F-OB to proteins, requiring additional time for SDS extraction in SDS-PAGE. Variability in protein-to-protein staining was observed, indicating potential differences in interaction between the dye and proteins.
1:Experimental Design and Method Selection
The study employed a commercial fabric fluorescent optical brightener (F-OB) for staining proteins on native and SDS-1D-PAGE. The F-OB was purified using a biphasic solvent system of dichloromethane and water. The staining efficiency was evaluated through absorption and emission spectra, mass spectrometry, and thin layer chromatography.
2:Sample Selection and Data Sources
Bovine serum albumin (BSA) and medium range molecular weight markers were used for standardization and sensitivity testing. Biological samples such as rabbit sera and rat lens γ-crystallins were also tested.
3:List of Experimental Equipment and Materials
Ranipal? (F-OB), Coomassie brilliant blue (CBB) R-250, molecular weight markers, BSA, bromo phenol blue, acrylamide, SDS, TEMED, 2-mercaptoethanol, Polyester silica Gel-G TLC plates, Sigma gel electrophoresis unit, Spekol 1200 spectrophotometer, Perkin-Elmer LS-3B fluorescence spectrophotometer, Agilent 1100 series LC/MSD trap, UVItec CCD-based digital image-based analysis system.
4:Experimental Procedures and Operational Workflow
Purification of F-OB, TLC analysis, spectral studies, mass spectrometry, polyacrylamide gel electrophoresis (native and SDS-PAGE), staining procedures (pre- and post-electrophoretic), and detection using CCD-based DIAS.
5:Data Analysis Methods
Fluorescent intensity was measured using CCD-based DIAS. Linearity of protein staining was analyzed by correlating the amount of protein loaded versus fluorescent intensity. Calibration plots were constructed based on linear regression analysis.
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