Purification of green fluourescent protein, GFP

Cell culture: 

Lysozyme solution Lysozyme, also known as muramidase or N-acetylmuramide glycanhydrolase, are glycoside hydrolases, enzymes (EC 3.2.1.17) that damage bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrin)

DNase I solution

Phenyl sepharose slurry and empty column 
The method for separation is hydrophobic interaction

   The bacterial cells’ outer membrane ruptures because the liquid inside the cell membrane will increase in the volume  and the cell wall cannot be tolerant this volumes.

The purpose of rupturing or lysing the bacteria is to separate the fluorescent proteins. These proteins attached DNA and other proteins in the cells.

After the last centrifugation, the pellet sill contains fluorescent proteins so it turns to green when lighted by UV. And the supernatant is green, indeed it contained FP. The reason for discard the pellet is that no need for further experiment, and i also contains broken cell membrane, proteins...

Hydrophobic interaction is based on the interaction between hydrophobic compounds. This lab is to purify the protein of interest, fluorescent protein.

a.       Equilibration buffer: 2 M NaCl the column at equilibrium state NaCl, water is absorbed on the

b.      Binding buffer: 4 M NaCl, increasing salt concentration makes protein more efficient to bind to hydrophobic surface of the column.

c.       Wash buffer 1M NaCl, wash some proteins have weak hydrophobic(more hydrophilic) interaction with column. and this cannot be used to elute FP.

d.      TE (elution) buffer , elute the most hydrophobic protein, fluorescent proteins. the water content will increase making FP difficult to attach to hydrophobic surface of the column. then FP will slowly be eluted. it takes about 15min to elute FP. 5 fractions-1mL are collected and tested protein assay or Using UV light to make it these fractions become green.

The buffer has the highest concentration will be used as binding buffer, and the one has the least will be the elution buffer.

Use the Coomassie based pierce test solution, the collected fraction turned to blue, or protein is proved to be separated by UV.