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Protein Dynamics in Living Cells

Protein Dynamics in Living Cells

Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...

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The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

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Published on: January 22, 2013

A highly sensitive near-infrared fluorescent detection method to analyze signalling pathways by reverse-phase protein

Laurence Dupuy¹, Christophe Gauthier, Guillaume Durand

¹BIOS group, INRA, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France.

|October 17, 2009

Summary

This study introduces an enhanced reverse-phase protein array (RPPA) detection method using near-infrared imaging and signal amplification. This improved technique allows for highly sensitive quantification of protein phosphorylation in cell signalling studies.

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Area of Science:

Biochemistry
Molecular Biology
Systems Biology

Background:

Quantitative analysis of protein phosphorylation is crucial for understanding cell signalling.
High-throughput methods are needed to map complex signal transduction networks.
Reverse-phase protein array (RPPA) offers potential for high-throughput analysis.

Purpose of the Study:

To develop an improved, highly sensitive detection method for RPPA.
To enable quantitative analysis of protein phosphorylation from small biological sample amounts.
To validate the method for studying intracellular signal transduction.

Main Methods:

Developed an enhanced RPPA detection protocol combining near-infrared imaging with tyramide signal amplification.
Tested the method's limit of quantification (LOQ) using serial dilutions.
Validated the method using follicle-stimulating hormone-triggered signalling in HEK293 cells.

Main Results:

Achieved a lower LOQ of 0.21 attomoles, a significant improvement from 6.84 attomoles with direct detection.
Consistently detected phosphorylated proteins in the sub-attomole range from <1 ng of total cell extracts.
Demonstrated excellent intra- and inter-slide reproducibility, correlating well with Western blot analysis.

Conclusions:

The enhanced RPPA method provides unprecedented throughput for analysing signalling network dynamics.
This technique allows for the capture of subtle regulatory events using minute biological sample quantities.
RPPA combined with amplified near-infrared detection is a powerful tool for systems biology in cell signalling.