Practically Science

While microscopic methods with protein and atom scale resolution – that don’t break the diffraction barrier! – exist (e.g. electron microscopy, atomic force microscopy, x-ray crystallography, etc.), they tend to be more practically difficult than fluorescence-based microscopy (see posts on Fluorescent Probes and Fluorescent Antibodies). Therefore, to give fluorescence microscopy nanometer resolution, several super resolution techniques have been developed that combine clever (1) optical/photophysical and (2) computational-processing tricks to “clean up” the “blurred data.”

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ELISA or Enzyme-Linked Immunosorbent assay is the most commonly used method for measuring proteins concentrations in solution. It is extensively used both in the laboratory (e.g. culture supernatant) and the clinic (e.g. blood tests) due to its simplicity and adaptability to other protein-based assays such as high throughput screening. The way it works is outlined in the figure above and described in detail below:

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Flow cytometry is a technique that can quantitatively measure (1) protein expression levels per single cell and (2) amounts of different cell-types (based on a protein-maker) for thousands of cells in minutes! Flow cytometers use hydrodynamic focusing to force a mixture of stained cells into a single-file line through a flow cell. Then, each cell, sequentially, passes through a laser beam which excites the fluorophore allowing quantification of protein expression for all proteins that are stained!

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Antibodies are immune proteins that can be easily engineered to bind (and “detect”) any given protein in via a simple vaccination method. To “signal” the presence of detected proteins, fluorophores are attached to “stain” the protein (and cell or tissue expressing it) a particular color. These fluorescent probes, have been utilized to identify the location of specific proteins in tissue sections (histology), single cells (microscopy) and quantify the amount of protein in cells (flow cytometry) and in complex mixtures (western blotting).

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