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

Antibodies have become a great tool for identifying and characterization of different proteins in different model systems using the technique of immunohistochemistry and Western blotting. The precision and sensitivity of these molecules allow us to develop a clear-cut picture of molecular cascades and signalling pathways. The technique of characterization of proteins by immunohistochemistry is very old, however it requires elaborate standardization and rigorous scientific approach to select the right antibodies for the experimental need.  

Protein purification based on fusing peptide affinity tag to recombinant protein is widely preferred due to its ease of use. Immobilized Metal Ion Affinity Chromatography (IMAC) was based on the principle of binding of specific amino acid side chains of a protein to metal ions immobilized on a matrix. Histidine amino acid has high affinity to metal ions, six histidine residues are fused to N-terminal or C-terminal end of a protein during cloning, and the protein obtained could be ~95% pure after passing through metal ion based resin. Protein purification based on metal affinity chromatography is used with all the expression systems including E.coli, Yeast, Insect cell and mammalian cell expression systems. 

Immunofluorescence is an essential and a powerful tool in molecular biology for the visualization of cellular structures, protein localization and intracellular processes. The resultant sample produced after immunofluorescence is analyzed using different microscopic methods such as confocal microscopy, epifluorescence, TIRF, or GSDIM depending on the experiment.

At a cellular level, there is always a homeostasis maintained between cell division and cell death. The process of growth and development involves an active balance of both of these mechanisms. Cell death occurs through three distinct mechanisms depending upon the molecular signal: Apoptosis, Necrosis, and Autophagy. All these processes have specific morphological and biochemical hallmarks that help them to get identified through different techniques.  Also, these processes are crucial in a normal cellular state, while a heightened or reduced rate of these processes lead to diseased conditions. Autophagy involves intracellular protein degradation and is an important aspect of cell starvation, aging, and differentiation. Apoptosis is also known as programmed cell death, where a strict tightly controlled event making apoptotic bodies to promote cell death. Necrosis results into the release of cellular components in the extracellular space after cell destruction.