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

The cardinal principle behind immunoprecipitation (IP) is the isolation of an antigen by interaction between the antigen and a specific antibody conjugated to a sedimentable matrix. Antigen source may vary from cells or tissues without labeling, radio labeled or tagged cells, labeled or unlabeled proteins isolated from subcellular fractionation or in-vitro translated proteins. Immunoprecipitation can also be helpful in protein fraction analysis separated by gel filtration or differential sedimentation based on density gradients.   In order to capture interacting antigens, antibodies developed from various animal species that could be either polyclonal or monoclonal, are used for immunoprecipitation. These antibodies are tagged  non-covalently with specific immunoadsorbents such as protein A–or protein G–agarose or covalently linked with a solid-phase matrix.

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: