SDS-PAGE

Techniques Paper About SDS-PAGE Technique Used In Biochemistry For Research And Clinical Purposes

Techniques Paper About SDS-PAGE Technique Used In Biochemistry For Research And Clinical Purposes

Introduction

The most widely used technique for electrophoresis separation of proteins in polyacrylamide gels is electrophoresis in the presence of SDS, so-called. SDS-PAGE. SDS (sodium dodecyl sulfate) is a strong anionic detergent, which destroys the protein structure of higher order, denaturing proteins to form a linear (primary structure) and gives them a net negative charge (Alberts, Bruce, et al, 2002). SDS-PAGE is the acronym in English of sodium dodecyl sulfate polyacrylamide gel electrophoresis (electrophoresis in gel of polyacrylamide with sodium dodecyl sulfate). It is a technique widely used in biochemistry, genetics, molecular biology and forensic science to separate proteins according to their electrophoresis mobility (depending on chain length polypeptide, molecular weight, posttranslational modifications and other factors) (Alberts, Bruce, et al, 2002). Thanks to most proteins SDS acquire a load / mass identical, so you get a fractionation due to: the difference in weight, chain length (size) and form of the protein. Electrophoresis is the method more widely used to analyze proteins (Alberts, Bruce, et al, 2002).

Discussion

SDS-PAGE electrophoresis

It is assumed that an anion dodecylosiarczanowy statistically falls into two amino acid residues. In addition to the distributed sample is added to strong reducing agents, such as 2-mercaptoethanol or DTT to reduce disulfide bridges present in proteins. It can, therefore, be assumed that the rate of protein migration during SDS-PAGE electrophoresis is dependent only on their mass and is directly proportional to its logarithm. Of course, there are proteins for which there are exceptions to this rule, and their migration is impaired (Alberts, Bruce, et al, 2002). These include proteins endowed with a high load native (e.g. histone proteins, migrating more slowly than it would point to their mass) and membrane proteins. However, for most proteins the rate of migration is dependent only on the weight. Therefore, it is possible to use the so-called, protein size standards (Alberts, Bruce, et al, 2002). Size standards are a mixture of a few or several proteins of known mass. After the parallel section along with the analyzed sample, it is possible to estimate the weight of test protein. For most of the protein error of estimating the size is between 5 to 10% (Campbell, Neil, 2004).

Chapter SDS-PAGE is conducted generally discontinuous gel technique. As a standard electrophoresis, buffer system is used tris-glycine. Ions involved in the thickening of the analyzed proteins in the gel are zatezajacym chloride anions and glicynianowe. Zatezajacego gel pH is slightly higher than the pI of glycine (Campbell, Neil, 2004). Under these conditions, most of the glycine is in the form of zwitterions having a net zero electric charge and not migrating in the electric field. Only a small part of them has at any given time the total negative charge and migrates towards the anode (electrode +). Glicynianowy anion migrates slowly so as so. "Ion delayed" (called trailing ion). While the anions chloride, having a high electrophoresis mobility, moving ...