《凝胶电泳》.docxVIP

GEL FILTRATION ●BACKGROUD 1、 Gel can filtrate proteins with its special characters of the space network structure , porosity and insolubility. Because proteins have different sizes, when proteins get through the gel grains they meet different resistance .Macromolecules can’t get in the gel grains ,and are completely excluded.They flow faster out of gel. Smaller molecules can diffuse freely and get in the grains,finally be taken away by the eluent.The smaller the molecules are ,slower they flow.Some molecules of medial size will get in the grains in part.The flow rate is also in the middle. 2、Several physical properties must be introduced to define the performance of a gel and solute behavior. Some important properties are: a. Exclusion Limit This is defined as the molecular mass of the smallest molecule that cannot diffuse into the inner volume of the gel matrix. All molecules above this limit elute rapidly in a single zone. The exclusion limit of a typical gel, Sephadex G-50, is 30,000 daltons. All solute molecules having a molecular size greater than this value would pass directly through the column bed without entering the gel pores. b. Fractionation Range Sephadex G-50 has a fractionation range of 1500 to 30,000 daltons. Solute molecules within this range would be separated in a somewhat linear fashion. c. Water Regain and Bed Volume（Vt） Gel chromatography media are often supplied in dehydrated form and must be swollen in a solvent, usually water, before use. The weight of water taken up by 1 g of dry gel is known as the water regain. For G-50, this value is 5.0±0.3 g. This value does not include the water surrounding the gel particles, so it cannot be used as an estimate of the final volume of a packed gel column. Most commercial suppliers of gel materials provide, in addition to water regain, a bed volume value. This is the final volume taken up by 1g of dry when swollen in water. For G-50, bed volume is 9 to 11 ml/g dry gel. d. Gel Particle Shape and Size