Summary And Review Of The Article Detecting Proteins By T. Rabilloud

This article is entitled “Detecting Proteins”. The article states as being a useful guide for choosing the correct stain, dye, florescent label, or radioactive label for proteins. It was published on January 1, 2000 on the “Analytical Chemistry” Journal by Thierry Rabilloud, while working with The University of Grenoble. Rabilloud is a CNRS senior researcher at CEA Grenoble. He is very interested in proteomics and 2-D electrophoresis. The article was published by “Analytical Chemistry” a peer-reviewed journal published since 1929 by the America Chemical Society.

Summary

In this article Rabilloud hypothesizes that there is not one correct way to properly label proteins. It all depends on certain factors that he refers to as “key issues” the detection of threshold, linearity, homogeneity, and reproducibility. He establishes that there are no supporting variables that state one technique is perfect for all needs. He himself has used many of these techniques during his job as a researcher in the Grenoble University. The experimental design used here are the different labeling techniques explained in this article. He explains the pros and cons of many labeling techniques that are used to bind to proteins like Covalent fluorescent labeling, and Radioactive labeling. He states the importance about attaching the label before IEF (Isoelectric focusing) which is performed under strongly denaturing environments so the noncovalent bonding will be disturbed.

Unfortunately, covalent labeling of proteins before IEF must meet certain requirements ruled by the chemistry of proteins and process of IEF. Because the number of active sites in a protein is limited, only very sensitive labels are introduced by covalent grafting. This limits the scope of labeling that can be done before IEF. However, using covalent labeling before the IEF gives you the possibility of multiplexing, which contains labeling lots of different samples with different probes. In covalent fluorescent labeling; intense light absorption, high quantum yield, and limited fading are additional chemical restrictions that further limit the number of labels. These types of labeling have many limited drawbacks like fluorescein derivatives, they can’t be used prior to IEF because of the fluorescent molecule carries a negative charge. In radioactive labeling, because of safety problems, it has to be done before IEF. Radioactive labeling can be very effective by introducing radioactive metabolites into the sample. Which doesn’t have any problems of pI modification because the amino acids themselves are labeled.

These are just a couple of the many labeling techniques mentioned in this article by Thierry Radilloud. The results of the information in this article are based on facts of his own experience while performing these techniques. He concludes that for dye staining the, maximum occurs when the dye is bonded to all the active sites that are available in the protein. For the best results you should keep the “key issues” in mind to help guide your search for the labeling technique that will work for what you need.