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

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.

Sputum Liquefaction: from viscous, heterogeneous mucus to equivalent, homogenous samples

Sputum processing has one main purpose; to render the viscous, heterogeneous specimen to a homogenous state (evenly distributed suspension). This homogenous suspension will be dispensed into multiple aliquots for cryopreservation, smear microscopy, gram stain, and culture. Each aliquot should be representative of the original specimen. This can be accomplished through the process of sputum liquefaction.

Reporter Gene Assays and their applications

Reporter gene assays are paramount for study of regulation of gene expression by gene regulatory elements (cis-acting factors), transcription factors or exogenous regulators (trans-acting factors). In reporter gene assays, the activity of a reporter gene is measured. A reporter gene is joined to a target regulatory DNA sequence in an expression vector, which is then transfected into the cell type of choice. The reporter gene is transcribed and translated in the cells and its activity is measured to access the strength or function of the target regulatory DNA sequence or study effects of transcription factors or potential drugs etc.