01:694:301 Lecture Notes - Lecture 3: Electrophoresis, Affinity Chromatography, Isoelectric Focusing
14 Nov 2018
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Molecules smaller than pores move quicker/easily than larger molecules in gel: when electric field applied, proteins migrate from negative to positive electrodes (top to bottom) Usually performed in slab of polyacrylamide gel (sds) Under denaturing conditions, proteins can be separated based on mass: first dissolve proteins in sds, then !-mercaptoethanol to reduce disulfide bonds, sds anions bind to aa residues; complex sds + denatured protein. Large net negative charge: after electrophoresis dye staining is used to help visualize protein movement. 3. 11: separation of proteins based on acidic/basic residues, ph gradient on gel; protein migrates until it hits ph = pi. Once protein reaches pi; electrophoretic mobility is zero stops. Thus proteins are separated horizontal direction basis of pi and vertical direction basis of mass: dialysis and gel filtration are separations on the basis of size, dialysis. Proteins can be separated from small molecules (i. e. salt)
