In this experiment we applied gel filtration of enzyme b using sephadex as column material. This is a chromatographic technique used for protein purification. The principle is based on fractionation of proteins based on the molecular weight or size of protein molecules. In this system when the mixture of the protein is applied at the top of the gel filtration column and washed with buffer solution, depending on molecular exclusion limit of column material, the large protein molecules are excluded from the internal volume and thus leave the column first. The smaller protein molecules are absorbed by the pores of the column material and leave the column later.
We collected 24 fractions from the column but 16 of them are illustrated at the data. Because the other fractions couldn’t measured correctly. From these data we calculated the protein content, specific activity, purification fold and total activity of each fraction. The results show that 4th and 6th fractions have the higher activity. Also the protein content and total activity versus fraction number graphic is plotted. At this graphs the first peak illustrates the first eluted fraction and this is probably eluted from the void volume also from the second graph we can see that the firstly eluted fractions activity are lower.
1. Which fraction is the most active?
– 4th and 6th fractions are most active since their total activity is equal to each other and higher than the other fractions.
2. Calculate specific activity, purification factor, % recovery.
– Calculations are done at the calculation section.
3. Which fraction has the highest purity?
– 11th fraction has the highest purity since it has the highest purification fold.