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

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. 

Reporters have contributed significantly to the field of biology and its progress. Genetic reporters are widely used in drug development, biomedical research, and biochemistry. In a nutshell, the reporter gene is fused with the gene of interest and cloned into an expression vector. The expression vector with the chimeric sequence is transferred into suitable cells. The expression of the gene in the cell is measured by the activity of the reporter protein. A textbook good reporter gene should have high sensitivity and can be measured quantitatively after expression.