The Protein Man's Blog | A Discussion of Protein Research

Tetrazolium reduction assays are characterized by reduction of various tetrazolium analogs (MTT, MTS, XTT, WST-1, and INT) and quantification of the subsequent color change. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was originally developed as an alternative to the radioactive thymidine incorporation assay. It was one of the first assays to be developed for the 96-well plate format intended for high throughput screening in 1983 by Tim Mossman. The MTT assay differs slightly from the radioactive thymine incorporation assay as the MTT assay measures the cell viability of a solution as opposed to cell proliferation. This assay is therefore is an incredibly useful tool in determining drug cytotoxicity or differentiating between multiple cell lines.

Protein Storage and Stability

What are Proteins?

As one of the multifarious macromolecules, proteins are complex and crucial for cellular functions. Proteins are polymers built of monomer subunits called amino acids connected by a specific type of covalent bond known as a peptide bond. The properties of the protein depend on the type of amino acids present in them. Although the primary structure of the protein comprises of the amino acid sequence, the functional properties of the protein crucially rely on the three-dimensional or tertiary structure. Similarly, protein modifications for e.g., glycosylation, phosphorylation, may change the properties and function of the protein. These modifications alter the local conformation and mediate folding or stability as well as drive proteins to different cellular compartments. Proteins also display remarkable variability in terms of structure and flexibility depending upon their folding patterns. Some proteins are relatively rigid hence can function as structural meshes or connective lines. Proteins with reversible conformational changes (polymerization or depolymerization) are crucial for protein-protein interaction, growth and the transmission of information from cell to cell or within the cell.

The BCA assay (bicinchoninic acid assay) developed by Paul K. Smith et al. has become one of the most widely used protein assays since its introduction in 1985. The advantages offered by the assay help to explain the popularity and widespread use over other protein assays. The advantages of the BCA include:

Purified and commercial antibodies are routinely stored in buffers that contain the proteins bovine serum albumin (BSA) and gelatin as these act as stabilizers during long term storage.  The presence of 0.2-1% (2-10mg/ml) BSA and/or gelatin help to stabilize antibody solutions that are less than 1mg/ml during long term storage.  For the majority of immunodetection techniques (ELISA, Western blotting, immunoprecipitation) the presence of the stabilizers do not interfere with these immunodetection techniques and can be used without clean up.