CHEM237L Chapter Notes - Chapter 4: Size-Exclusion Chromatography, Aromatic Amino Acids, Coomassie Brilliant Blue
12 Jul 2017
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Size exclusion column to separate and inspect eluted fractions for catalase and myoglobin through assays and absorbance while running an sds. Page and calculating molecular weight, net charge, molar extinction coefficient, rf, and protein concentration. Size exclusion/gel filtration chromatography: proteins have different: charge, hydrophobicity, size, binding: ligand affinity, column beads have pores with a defined size that can be made of polyacrylamide cross lined with n,n" methylene-bis-acrylamide (bio gel. Smaller proteins: fit through bead pores, go through column slower (will elute within the fractionation limit) Larger proteins: too bit to fit through pores, elute faster (than upper fractionation limit: size exclusion resins are characterized by their fractionation range = molecular weight range (protein size/mw) that a resin can separate. Void volume: volume of buffer that surrounds the resin beads can be found by measuring the total volume displaced once the molecule > upper fractionation limit elutes from the column.
