Reference no: EM133300113

Using gel filtration chromatography (GFC), a matrix was selected with an exclusion limit of 1000 kD (effective separation range = 10-1000 kD). A tissue homogenate sample was placed on the column with the hopes of effectively separating 3 proteins into separate fractions. The 3 proteins of interest were catalase (240 kD), glutathione S transferase (45 kD) and glutathione peroxidase 4 (22 kD). The sample was added to the column and eluted with appropriate buffer. It required 30 ml of elution buffer to elute all the protein and biomolecules from the column and the researcher made a mistake and collected only three 10 ml fractions. This was a mistake because the fraction collector was set from a prior experiment. Given this mistake, there were only 3 fractions collected: an early eluting fraction, including the void volume (A), a middle fraction (B) and a much later eluting fraction (C).

Would this column effectively separate GST and glutathione peroxidase 4 as the experiment was carried out? If you think it would not, what would you change in the existing experiment to effectively separate the proteins? You have no other columns in the lab so what could you change about the way this experiment was carried out?