TCI America 6-Aminohexanoic Acid [for Biochemical Research] A2255

6-Aminohexanoic Acid [for Biochemical Research] /
In this section, proteases are categorized into three modes of actions: (1) serine protease; (2) cysteine protease; (3) metalloprotease.1)
Serine proteases are inhibited by AEBSF, benzamidine, etc.2) Cysteine proteases are inhibited by compounds which react with SH groups such as 2-iodoacetamide or 2-iodoacetic acid.3) Metalloproteases are inhibited by chelating agents, EDTA or 1,10-phenanthroline.4)
In the course of protein extraction, proteolysis is considered to be a major problem because it leads to decreasing yields. Addition of protease inhibitors helps to avoid the proteolysis and improves recovery of the desired protein.5) In the experiment of immunoprecipitation, protease inhibitors are also used to avoid decomposition of antigens or antibodies by proteolytic impurities.6) Protease inhibitors which are frequently used in biochemical research are illustrated in this section.
Thiol protease inhibitors, 2-iodoacetamide and 2-iodoacetic acid, cannot be used in protein extraction, because they form covalent bonds to the SH group of the proteins. In protein research, they are used as an alkylation reagent to derivatize the proteins which are extracted from an electrophoresis gel for mass spectrometric analysis.7)

6-Aminohexanoic Acid [for Biochemical Research] /
Electrophoresis is a technique which separates charged biomolecules based on the rate at which they migrate in an applied electrical field. In many cases, electrophoresis of proteins are performed using polyacrylamide gel electrophoresis (PAGE).1) For molecular weight estimation and purity determination of proteins, sodium dodecyl sulfate (SDS)-PAGE is frequently employed. SDS is a strong denaturant of proteins and is added to samples, gels, and buffer solutions for electrodes when proteins are separated with electrophoresis. As SDS not only denatures protein but also binds to the protein, when SDS is used in conjunction with a reducing reagent such as 2-mercaptoethanol to cleave disulfide bonds in the protein, and the protein is completely denatured, the amount of SDS bound is almost always proportional to the molecular weight of the protein. Resultantly, the protein is negatively charged. Therefore, the denatured protein can be separated by molecular weight independently of its structure and biological properties.
Laemmli's method is the most widely used system of SDS-PAGE.2) In this method, the separation and the stacking gel contain Tris-HCl and the upper and lower buffer reservoirs contain Tris-glycine. All components of the system contain SDS. The advantage of Laemmli's method is that it gives sharper bands in the final plate.1)