Exp.5 Electrophoresis

ELECTROPHORESIS

Electrophoresis 1. Definition ~ is the forced migration of charged particles such as macromolecules, in an electric field. Cations move toward the cathode and anions move toward the anode.

Macromolecular Charges in Solution  Protein,

amino acid;  Nucleic acid (DNA,RNA)

 Isoelectric

point

the particular pH at which the macromolecule is electrically neutral.

2. Principles of Electrophoresis E fv

qE

net force Ｆ = Ｅ q  frictional force ｆ =f ｖ Because net force= frictional force So ｖ = Ｅ q/ f 

Electrophoretic mobility U (an intrinsic property )

v q U= = . E f q (decided by Charge) Charge varies as a function of building block molecule composition and buffer pH .





f

based on Size (decided by molecular weight), Shape

3. Impact Factors of Electrophoresis 3.1 Properties of the Molecules 





Isoelectric point (Molecular charge) different charges and strengths in buffer. Molecular Shape a long, loose protein travels at a slower rate than a globular protein in a gel. Molecular weight (Molecular Size) A smaller molecular has a faster U.

2. Properties of the electrophoretic System  Electric field strength E  Property of the support medium influence separation in 3 aspects: restrictions on mobility; effect on diffusion ; electroendosmosis. common zonal support media : paper ; cellulose acetate; agar or agarose; starch ;polyacrylamide

 1. 2.

Role of buffer Solution pH ; Ionic Strength ;

I=

1 2

∑c z

2 i i

,

at ion concentrations of above about 10 - 100 mM, the macromolecular charges are fully screened and there are no electrical interactions with other large molecules.

Detection of components after electrophoretic separation 1.Proteins Special protein dye:

Amino Black-10B; Coomassie brilliant Blue R-250 or

G250; Silver staining;

Radiolabelled Proteins (C-14, H-3, P-32) Immunochemical detection: Western Blotting

2. Glycoproteins Schiffs Reagent (Neutral Glycoprotein); Alcian blue (Acidic mucopolysaccharides) 3. Lipid Sudan black 4. Enzymes Appropriate enzymatic methods 5. Nucleic acids Ethidium Bromide or SYBR gold