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

Nature has ability to generate an unending array of molecules from very small number of building blocks, even simple microbes such as E. coli have synthetic chemical prowess that can bring the most sophisticated organic chemistry labs to their shame. Inspired by the nature’s ability to knit complex molecules from a small number of building blocks, using simple modular reactions, Berry Sharpless, and his coworkers, defined and invented the area of ‘click chemistry’. Together, Berry Sharpless and M. G Finn redefined the use of simple modular reactions that had already existed to generate new ‘functions’ or properties and termed it as ‘click chemistry’.

Protein assays are one of the most widely used scientific methods, particularly in life sciences. An assay is an efficient spectroscopic procedure that analyzes the concentration of protein in a solution, also referred to as protein concentration quantitation. Being able to measure this concentration is an integral part of any laboratory workflow involving protein extraction, purification, labeling, or analysis. It is necessary for an array of research, including processing protein samples for isolation, separation, and analysis by chromatographic, electrophoretic, and immunochemical techniques. There are different types of assays and each can be useful for different applications, but many assays work in similar ways and utilize standards.

With so many protein assays to choose from, each with its own advantages and limitations, you have to consider their suitability for the application. In some instances, it can be necessary to utilize more than one type of assay. Here, we discuss how to select the most appropriate protein assay.

1-Fluoro-2,4-dinitrobenzene (DNFB), also known as Sanger’s reagent, was first used by Sanger to detect free amino acids of Insulin. DNFB undergoes nucleophilic aromatic substitution with the N-terminal amino group of a peptide or protein. After hydrolysis of the peptide or protein, the individual amino acids separate and only the labeled N-terminal amino acid can be detected by a colorimetric detection at specific wavelength. DNFB is hence used in protein sequencing to determine N-terminal amino acid.