Experiment 4 (Day 5): Further Questions

The answers below adress the questions in our practical manual.

1) Plot a graph of your A476 absorbance values (Y axis) vs fraction number. Comment on your chromatogram.

Refer to the discussion section in Experiment 4 (Day 5).

2) GFP has Mr (molecular weight) around 27, 000 kD. Though we were unable to see them, the cell extract also contained hundreds or even thousands of other proteins. Do you think a protein with a Mr of 50, 000 kD would elute in a fraction before or after GFP? Why or why not?

A protein with a Mr of 50, 000 kD would elute in a fraction before GFP, this is because it is bigger than GFP. Therefore larger protein, which fit in fewer pores, takes a more direct path through the column and elute first. The smaller GFP are slower to elute as they meander in and out of pores spending less time in the liquid mobile phase of the column.