Green Fluorescent Protein (GFP) Purificaiton

1. Green Fluorescent Protein (GFP) Purificaiton

2. Recombinant Cell

3. Protein Purification Process - Phase 1 • Isolate colonies of bacteria and grow in liquid media (nutrient broth in incubator for 2 days- shake often!) • Centrifugation – separate bacteria cells from liquid media by spinning • Heavier bacterial cells will fall to bottom of test tube a create a PELLET of bacteria – pellet will glow in the dark! • Liquid media will remain on top as SUPERNATANT (this is to be discarded)

4. Protein Purification Process – Phase 1 3. Resuspend bacteria pellet in buffer solution a. Add the buffer solution to the test tube b. Quickly pipette the solution multiple times in the test tube to mix and resuspend the bacteria – no clumps of bacteria should be observed 4. Add lysozyme to breakdown bacterial cell walls. 5. Freeze cells to rupture cell walls

5. Protein Purification Process- Phase 2 6. Centrifuge the lysed bacteria a. Larger cell materials, like cell wall/membrane, will fall to bottom and become pellet b. Smaller proteins, including GFP, remain in the supernatant (supernatant will glow)

6. Protein Purification Process- Phase 3 Protein Chromatography – a technique used to separate proteins in a mixture Three steps: 1. Binding 2. Wash 3. Elution

7. Protein Purification Process- Phase 3 • In chromatography, a cylinder, or column is densely filled with a "bed" of microscopic beads. • These beads form a matrix through which proteins must pass before being collected. • The matrix the has an "affinity" for the molecule of interest (GFP), but not for the other bacterial proteins in the mixture. • GFP "sticks" to the columnallowing it to be separated from the bacterial contaminants.