SDS-Polyacrylamide Gel Electrophoresis (PAGE)

, experimental purpose:
1. Learn the principle of
SDS
-PAGE separation
protein

;II, experimental principles:
1, electrophoresis:
(1) definition: refers to the charged particles in the electric field in the direction of the electrode opposite to the charge they themselves.
(2) Factors affecting electrophoresis effect:
(1) size and shape of charged particles: the larger the particles, the slower the electrophoresis speed, the faster the reverse
; The slower the speed, the faster the
(3) solution viscosity: the greater the viscosity, the slower the electrophoresis speed, and the faster the
(4) solution pH: affects the dissocation of the separated substance, Closer to the isoelectre, the slower the electrophoresis speed, the faster the reverse;
(5) electric field strength: the smaller the electric field strength, the slower the electrophoresis speed, the faster the opposite;
(6) ion strength: the greater the ion strength, electrophoresis The slower the speed, the faster the reverse;
(7) electroplysis phenomenon: the relative movement of the liquid relative to the solid support in the electric field,
(8) the size of the support sieve: small aperture, slow electrophoresis, and the other is fast. 2, SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
(1) definition
polyacrylamide gel electrophoresis (PAGE): is a polyacrylamide gel as a carrier of a regional electrophoresis.
SDS-PAGE: The role of SDS (sodium dioxane sulfonate)
(2) SDS is introduced into the polyacrylamide gel system
SDS is an anion descaling agent that binds to proteins to form SDS-protein complexes. Because SDS carries a large negative charge, like a protein wearing a negatively charged "coat", the charge on the protein itself is masked, eliminating the difference in charge between protein molecules. Therefore, in electrophoresis, the migration speed of protein molecules mainly depends on the size of protein molecules
(3) SDS-PAGE classification:
3/4SDS-PAGE according to buffer pH and gel aperture differences into two categories:
continuous system: electrophoresis system buffer pH and gel concentration is the same, with electro-particles in the electric field, mainly by electrical particles and molecular effect.
discontinuous system: buffer ion composition, pH, gel concentration and potential gradient are not continuous, charged particles in the electric field swimming not only have charge effect, molecular sieve effect, but also has a concentration effect, so its separation strip clarity and resolution are compared to the former The
(4) polyacrylamide gel is produced:
polyacrylamide gel is polymerized by acrylamide monomer (Acr) and N,N'-fork diacrylamide (Bis) under the action of a catalyst. When there is a free base, Acr and Bis are aggregated.
there are two ways to trigger the production of free fundamentals:
(1) chemical method
(2) photopolymer legal (1) chemical polymerization:
trigger is ammonium persulphate (AP), catalyst N, N, N', N-tetramethyl ethyl diamine (TEMED), its base catalytic AP produces oxygen free agent, activated monomer to form free agent, polymerization occurs. The gel aperture formed by chemical polymerization is small and repetitive, which is used to prepare the separator,
(2) photopolymer: the
catalyst is the nucleolutin (VB
2
), in the presence of trace oxygen, the nucleotin photolyte forms a colorless base, the colorless base is oxidized into a free base by oxygen, and activates monomer polymerization. The gel aperture formed by light polymerization is large and unstable, which is suitable for the preparation of concentrates with large aperture.polymerization reaction:
(5) Polyacrylamide gel structure features:
(1) The basic structure of polyacrylamide is a long chain of acrylamide monomers, chains and chains are linked by a fork bridge The vertical and horizontal
(2) chain is staggered to form a three-dimensional mesh structure, which makes the gel have the nature of molecular sieve, and the
(3) mesh structure can also limit the diffusion movement of samples such as proteins, so that the gel has anti-convex effect, .