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A Quantitative Microscopy Technique for Determining the Number of Specific Proteins in Cellular Compartments
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A Quantitative Microscopy Technique for Determining the Number of Specific Proteins in Cellular Compartments
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Journal Article
A Quantitative Microscopy Technique for Determining the Number of Specific Proteins in Cellular Compartments
2011
Overview
This protocol describes a method to determine both the average number and variance of proteins in the few to tens of copies in isolated cellular compartments, such as organelles and protein complexes. Other currently available protein quantification techniques either provide an average number but lack information on the variance or are not suitable for reliably counting proteins present in the few to tens of copies. This protocol entails labeling the cellular compartment with fluorescent primary-secondary antibody complexes, TIRF (total internal reflection fluorescence) microscopy imaging of the cellular compartment, digital image analysis, and deconvolution of the fluorescence intensity data. A minimum of 2.5 days is required to complete the labeling, imaging, and analysis of a set of samples. As an illustrative example, we describe in detail the procedure used to determine the copy number of proteins in synaptic vesicles. The same procedure can be applied to other organelles or signaling complexes.
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