Topics: Protein Purification, Western Blotting, Protein Electrophoresis, Protein Estimation, Sample Clean Up, Protein Concentration, Protein Fractionation, Protein Extraction, Buffers & Chemicals, Protein Detection
Reactive oxygen species (ROS) are highly reactive molecules and free radicals formed as a natural byproduct of oxygen metabolism. These molecules, which are present at low levels in normal cells, play a vital role in maintaining healthy redox processes and ensuring proper function in the cells. However, an overproduction of ROS may cause oxidative stress, cellular damage, and DNA damage, which may lead to several physiological and pathological conditions.
Western blotting or immunoblotting is an indispensable technique, almost every published paper in area of molecular cell biology uses western blotting for detecting specific proteins in a sample of tissue homogenate or cell lysates. Western blotting combines resolving power of polyacrylamide gel electrophoresis (PAGE) or SDS-PAGE and specificity of antibodies to detect target proteins. Proteins are resolved on the basis of their molecular weight in SDS-PAGE and transferred from the polyacrylamide gel onto the membranes (Nitrocellulose or PVDF), which creates an exact replica of the protein separation pattern on the membrane. After transferring the proteins to the membrane, the membrane needs ‘blocking’ to ‘block’ non-specific binding sites on the surface of the membrane. Blocking is usually performed with Bovine Serum Albumin, Skimmed milk or purified milk Casein.
Poly-histidine tagged proteins account for more than 90% of routine recombinant protein expression and purification. Histidine tag has many advantages over other affinity tags such as small size, high affinity for binding to IMAC resins (Immobilized metal affinity chromatography) and allows one step purification of recombinant protein. But sometimes a histidine tagged protein doesn’t bind to the affinity column and evades it’s capture and subsequent purification. Although such instances are not very common but can be very frustrating whenever they happen. However, there are ways to troubleshoot if such a problem arises and the solutions are discussed below in a step by step manner.