研究目的
To develop a novel method for the gel-based in vitro analysis of the phosphorylation status of proteins using a TAMRA-labeled Phos-tag derivative for simple and rapid fluorometric staining.
研究成果
The TAMRA–Phos-tag staining method provides a simple, rapid, and quantitative approach for analyzing the phosphorylation status of proteins in gel-based assays, applicable to both phosphatase and kinase reactions. It offers advantages in terms of procedure simplicity, pH neutrality, and time efficiency.
研究不足
The fluorescence response per phosphate group varies among proteins, potentially due to intermolecular fluorescence quenching or steric hindrance, which may affect quantitative comparisons between different phosphoproteins.
1：Experimental Design and Method Selection:
The study utilized a TAMRA-labeled Phos-tag derivative for selective staining of phosphorylated proteins in polyacrylamide gels. The method involved three buffer solutions for staining, washing, and dilution.
2：Sample Selection and Data Sources:
Phosphorylated proteins (ovalbumin and β-casein) and nonphosphorylated proteins were used to evaluate the selectivity and sensitivity of the staining method.
3：List of Experimental Equipment and Materials:
Included a fluorescence imaging scanner with a 532-nm excitation laser and a 580-nm longpass emission filter, SDS-PAGE apparatus, and various chemicals for buffer preparation.
4：Experimental Procedures and Operational Workflow:
The staining protocol involved three steps: staining with TAMRA–Phos-tag, washing, and dilution, completed in less than 2 hours.
5：Data Analysis Methods:
Fluorescence intensities were quantified using densitographic software to analyze the phosphorylation status of proteins.
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