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

Recombinant proteins are usually expressed with an affinity tag that facilitates binding of the protein to an affinity matrix and results in high yield purification of proteins (usually above 90% yield) by exploiting avidity between the tag and affinity matrix. Tags are usually small peptides or proteins that facilitate selective binding to an affinity matrix. There are many types of tags available to facilitate protein purification, some common examples are poly-histidine tag, GST tag, FLAG tag. The bound proteins can be eluted using a chemical agent usually known as eluent that has a higher affinity for affinity matrix and results in the release of bound tagged proteins from the affinity matrix by chemically competing against the bound proteins for the binding sites on affinity matrix.

Transport tunnels in enzymes: a novel avenue for protein engineering to improve or modify the catalytic function

  In a previous blog article, we discussed western blotting technique and advancement in Western blot detection using fluorescence, not only fluorescence allows detection of multiple protein bands on the same blot (multiplexing) but is a more sensitive and reliable method of detection. The linear dynamic range of chemiluminescence is 10 times smaller than Fluorescence. This blog article focuses on one of the most useful and widely used applications of Western blotting, the quantitative western blotting. Quantitative western blotting combines western blotting with digital image analysis and allows molecular cell biologists to measure and quantify changes in protein abundance and modifications (Proteolysis and post-translational modifications).