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

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.

1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril (Chloroglycoluril), also known as Iodo-Gen®, is one of the best reagents used for iodination of proteins, hormones, antibodies, viruses, cell membranes, etc. This method was first developed by Fraker et al. This method of iodination with Iodo-Gen® is simple and inexpensive. Iodo-Gen® is water-insoluble and hence used in solid-phase iodination. Iodo-Gen® is used to incorporate radioactive iodine in aromatic amino acids like tyrosine. It iodinates proteins in oxidizing conditions, so there is no need for using excessive reducing agents that affects protein stability. The proteins are not directly subjected to oxidation in this method as the reagent sticks to the wall of the tube and protein is in solution. Either Iodo-Gen® is coated on the walls of glass vials or on polystyrene beads. Iodo-Gen® is dissolved in an organic solvent and the organic solvent is removed from the vial by using nitrogen. The Iodo-Gen® that sticks to the wall of the vial or beads later iodinates proteins in solution. Protein can be separated easily from unreacted reagent as Iodo-Gen® is water-insoluble.