Question

9. The following proteins were identified in a mixture. protein te precipitate MW (kDa) 4.8 22 IMRSO) 45% 80% 65% 20% 30% 45% 5.3 6.8 9.50 un 115 5.3 th(cont.) a) What is meant by "salting out" a protein using MgSO.? What happens on a molecular level? b) Draw a gel indicating proteins 1-6 order of elution in SDS-PAGE. Label where sample would start so I am clear on the validity of your answer. c) Draw their order of elution off of a a cation exchange column. d) Draw their order of elution off of a size exclusion column

Answer 1

9a Salling out is a purification methoel that relies on the basis of protein solubility Proteins are less solution soluble in solutions of high salt concentration because addition of salt sheilds, proteins with multi-ion chargest At Boelectric point net charge of protein is zero, and therefore it has least repulove forces and attractive force predominate This results in aggregation and precipitation. 6) SDS-PAGIE Separates Protein M Parates Proteins in mixture by their molecular in an applied electric field. When current is applied, all SDs- beund protein in a sample will migrate get would positively charged elechode. (-) Cathede Movement from cathode to anode Who is to 1 tl Anode (6) Cation exchange column Protein at pH LPI will be positively charged and at pH > BI will be negatively charged. Cation exchange column will tined to positively charged protein and elule off negatively charged particle .. Therefore, protein with higher po comes out last Order of elusion, 1 > 2 > 3 > 4 > 5 Protein 1 comes out first and protein 5 comes out last-

VEL (d) size exclusion column. "In size exclusion column, there are porous beads. smaller protein enter inside this beads interact with them and elite slowly. While larger proteins comes out faster. Order of elusion, 6 > 4 >5 > 1 2 3 Protein to come out first became it was higher molecular weight while protein 3 with lower molendar weight comes out last