Protein Purification.ppt

Protein Purification Molecular weight Charge Solubility Affinity Molecular Weight Ultracentrifugation Dialysis Gel filtration SDS PAGE Molecular Weight The lab in week 6 and 7 will involve separating a protein mixture by molecular weight using 2 methods: gel filtration and SDS PAGE Gel filtration separates by the native molecular weight SDS PAGE separates by the subunit molecular weight Gel Filtration This method relies on a column of beads of a specified pore size. This is known as a molecular sieve. Proteins (and other macromolecules) above a certain cut-off size cannot fit into the pores and so migrate down the outside of the beads. They will elute first. Smaller molecules below the cut-off can permeate the pores and so take longer to travel down the column. An elution profile Gel-filtration of the protein mixture. 1.2 ml of protein mixture (10 mg/ml) was loaded onto a 25 cm X 2.5 cm diam. Sephadex G-50 column equilibrated with buffer (50 mM Tris HCl, pH 7.5). The column was eluted with buffer at ~1 ml/min, collecting 2.5 ml fractions. The absorbance of each fraction was measured at 280 nm and 410 nm. SDS PAGE This technique involves loading a sample of your mixture onto a polyacrylamide gel (PAGE). Polyacrylamide works like agarose except the matrix has smaller pores and so polyacrylamide gels separate smaller molecules (like proteins). Agarose is used for much larger molecules such as DNA and RNA. SDS PAGE Unlike DNA and RNA proteins do not have a nice constant charge to mass ratio and can have any charge at a given pH, depending on their sequence, hence pI. To overcome this problem proteins are coated with a detergent, SDS, which makes them negatively charged. They then separate by molecular weight. SDS PAGE They then separate by molecular weight. The SDS will disrupt the secondary, tertiary and quaternary structure so the subunits will separate. For this reason SDSseparates by subunit molecular weight. SDSCatalase, cytochrome C, a-lactalbumin Hemoglobin, Cy