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ONE-STEP ANALYSIS OF PROTEIN COMPLEXES IN MICROLITER CELL LYSATES BY FLUORESCENCE CORRELATION SPECTROSCOPY

ONE-STEP ANALYSIS OF PROTEIN COMPLEXES IN MICROLITER CELL LYSATES BY FLUORESCENCE CORRELATION SPECTROSCOPY

(Summary description)The team of Roland Brock , Institute of Intercultural Cell Biology, Department of Molecular Biology, University of Tübingen, Germany, applied fluorescence correlation spectroscopy ( FCS & FCCS ) and immunofluorescence labeling to quantitatively analyze the interaction of endogenous protein molecules in micro-cell lysates . Traditional experimental techniques for studying endogenous biomolecular interactions (such as co-immunoprecipitation; Co-IP ) are cumbersome and tend to ignore weak molecular interactions. In response to this key point and difficulty in scientific research, the author first expressed the fusion protein ZAP-70-YFP in mouse 3a9 cells, then treated the cells with vanadate ( PV ), and verified the fusion protein ZAP-70 using FCS/FCCS technology -YFP can bind to the membrane protein CD3ε on the surface of the receptor . In order to exclude false positive results that may be caused by exogenously expressed proteins, the author used specific antibodies to fluorescently label endogenous proteins. The results of FCS/FCCS experiments showed that tyrosine kinase ZAP-70 could interact with CD3ε under PV stimulation. interaction. In order to further study the signaling mechanism of endogenous proteins after PV stimulation in cells, the authors used FCCS technology to study untransfected mouse 3a9 and human Jurkatcells; the results showed that: in 3a9 cells, the signal protein GRB2 and PLC γ 1 interacted with the scaffold protein LAT ( +PV ); in Jurkat cells, GRB2-LAT did not detect the interaction, but LAT-PLC γ 1. There is an interaction between SLP -76-PLC γ 1 ( +PV ). Finally, the author applied the demonstration of the above new technology to the screening of peptide drugs in cell lysate samples. This paper demonstrates a new experimental technique that only needs to add primary antibody and fluorescently labeled secondary antibody to a small amount of cell lysate, and then apply fluorescence correlation spectroscopy to quickly and quantitatively analyze molecular interactions. Compared with traditional techniques (such as CO-IP ), the FCS/FCCS method has the advantages of less sample volume, simple operation, and no need for sample purification, which is of great significance to basic biological research and drug screening.

ONE-STEP ANALYSIS OF PROTEIN COMPLEXES IN MICROLITER CELL LYSATES BY FLUORESCENCE CORRELATION SPECTROSCOPY

(Summary description)The team of Roland Brock , Institute of Intercultural Cell Biology, Department of Molecular Biology, University of Tübingen, Germany, applied fluorescence correlation spectroscopy ( FCS & FCCS ) and immunofluorescence labeling to quantitatively analyze the interaction of endogenous protein molecules in micro-cell lysates . Traditional experimental techniques for studying endogenous biomolecular interactions (such as co-immunoprecipitation; Co-IP ) are cumbersome and tend to ignore weak molecular interactions. In response to this key point and difficulty in scientific research, the author first expressed the fusion protein ZAP-70-YFP in mouse 3a9 cells, then treated the cells with vanadate ( PV ), and verified the fusion protein ZAP-70 using FCS/FCCS technology -YFP can bind to the membrane protein CD3ε on the surface of the receptor . In order to exclude false positive results that may be caused by exogenously expressed proteins, the author used specific antibodies to fluorescently label endogenous proteins. The results of FCS/FCCS experiments showed that tyrosine kinase ZAP-70 could interact with CD3ε under PV stimulation. interaction. In order to further study the signaling mechanism of endogenous proteins after PV stimulation in cells, the authors used FCCS technology to study untransfected mouse 3a9 and human Jurkatcells; the results showed that: in 3a9 cells, the signal protein GRB2 and PLC γ 1 interacted with the scaffold protein LAT ( +PV ); in Jurkat cells, GRB2-LAT did not detect the interaction, but LAT-PLC γ 1. There is an interaction between SLP -76-PLC γ 1 ( +PV ). Finally, the author applied the demonstration of the above new technology to the screening of peptide drugs in cell lysate samples. This paper demonstrates a new experimental technique that only needs to add primary antibody and fluorescently labeled secondary antibody to a small amount of cell lysate, and then apply fluorescence correlation spectroscopy to quickly and quantitatively analyze molecular interactions. Compared with traditional techniques (such as CO-IP ), the FCS/FCCS method has the advantages of less sample volume, simple operation, and no need for sample purification, which is of great significance to basic biological research and drug screening.

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