Photosynthesis Lab Report

The starting material for this lab was the dialyzed sample (stored at -20ᵒ C) from the previous lab. The CM sephadex resin (taken in a 50 mL tube) was already made swollen using Buffer C (20 mM HEPES, pH 7.9; 1 mM EDTA; 50 mM KCl). The dialyzed sample was thawed to the room temperature and gently poured over the resin. The tube was capped and kept on a rocker at room temperature for 1 hour. The tube was then centrifuged in a HS-4 rotor at 2500 rpm (1200g) for 5 minutes at 4ᵒ C. Supernatant was discarded and the column was washed twice (i.e. centrifuged); first in 30 mL of low salt Buffer C (20 mM HEPES, pH 7.9; 1 mM EDTA; 50 mM KCl), followed by 30 mL of high salt Buffer C (20 mM HEPES, pH 7.9; 1 mM EDTA; 0.2 M KCl). Each time after adding the buffers, PMSF (protease inhibitor) was added to the final concentration of 1 mM and the tube was kept on a rocker for 30 minutes at room temperature.
Each time after the first centrifugation as well as next two washes (Buffer C with low and high salt), the tube was kept still on a stand to allow the beads settle