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

Accurate protein quantitation is an integral part of any laboratory workflow involving protein purification, electrophoresis, cell biology, molecular biology, and other research applications. Careful consideration must be taken when choosing the appropriate protein quantitation assay for a specific protein sample. There are many protein assays to choose from, but all protein assays are adversely affected by interfering substances (such as detergents, reducing agents, and chaotropic agents) which are often used in the preparation of protein samples. To overcome the limitations of these interfering substances, G-Biosciences’ developed a proprietary Universal Protein Precipitating Agent (UPPA™) for use with their CL (Compatible Lowry) Protein Assay.

Chromatography is a versatile field with a wide range of applications. It’s accomplished by fractionating a mixture into its molecular components. Chromatography was first used in 1901 by Russian botanist Mikhail Tsvet when he realized the technique could separate plant pigments. It has since become widely developed and utilized for separation analysis in various scientific fields.

Membrane proteins play a key role in cellular processes including transport of molecules, signal transduction, utilization of energy and maintenance of cell and tissue structures. It has been determined by genome sequencing that around 30% of genes encode membrane proteins. Furthermore, they are pharmacologically significant as 50% of the current drugs target the membrane proteins.  It is therefore of utmost importance to isolate membrane receptor complexes in functional active form for functional and structural studies, crystallization etc. However in spite of their significance, knowledge of structure and function of membrane proteins is lagging behind soluble proteins due to hurdles like low abundance and their isolation in native form from biological membrane. The obstacle of low abundance of membrane protein can be overcome with heterologous expression of these proteins and employing techniques similar to expression of soluble or cytosol proteins. Isolation of membrane proteins from biological membrane is carried out by solubilization and this process needs fine-tuning in order to purify functionally active membrane receptors complexes.

Biotin, a 244 Dalton molecule, exhibits an extraordinary binding affinity for avidin and streptavidin (K_a=10¹⁵ M^-1). This high binding affinity means that proteins, particularly antibodies, are routinely labeled with biotin.  The labeled proteins can now be rapidly purified with avidin and streptavidin resins or detected with streptavidin and avidin coupled to enzyme reporters, such as HRP (horseradish peroxidase.