SDS polyacrylamide gel electrophoresis for N-terminal protein sequencing.

Abstract

AbstractPolyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) is a very common technique used for analysis of complex mixtures of polypeptides. It has great resolving powers, is rapid, and is suitable for proteins of either acidic or basic pI. The last is because the protein is reacted with SDS, which binds to the protein in the approximate ratio 1∶4∶1 (SDS:protein, w/w) and imparts a negative charge to the SDS-protein complex. The charged complexes move toward the anode when placed in an electric field, and are separated on the basis of differences in charge and size. SDS-PAGE is commonly used to estimate proteins’ molecular weights, but estimates are approximate (being termed “apparent molecular weights”) and sometimes prone to marked error. For instance, disproportionally large increases in apparent molecular weight may occur upon covalent phosphorylation of a protein (1), or artificial entrapment of phosphoric acid (2). Most designs of SDS-PAGE employ a “stacking gel.” Such a system enables concentration of samples from comparatively large volumes to very small zones within the gel, thus giving narrow bands of the different proteins, which are then better resolved. The principle involved in this protein concentration (or “stacking”) is that of isotachophoresis. It is set up by making a stacking gel on top of the “separating gel,” which is of a different pH. The sample is applied at the stacking gel and when the electric field is applied, the negatively charged complexes and smaller ions move toward the anode. At the pH prevailing in the stacking gel, protein-SDS complexes have mobilities intermediate between the faster Cl- ions (present throughout the electrophoresis system) and the slower glycinate ions (present in the cathode reservoir buffer). The protein-SDS complexes concentrate in the narrow zone between Cl- and glycinate ions. When the moving zones reach the separating gel with its different pH, their respective mobilities change and glycinate overtakes the protein-SDS complexes, which then move at rates governed by then size and charge in a uniformly buffered electric field. Isotachophoresis is described in more detail in the literature (3).KeywordsApparent Molecular WeightAmmonium PersulfateAnalar GradeAcrylamide MonomerReservoir BufferThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.