Pre-Column Work

Prepare a plasmid prep-pET24a(+) cut NdeI+EcoRI with Tn insert cut NdeI+EcoRI,

Day 1

Transfect the plasmid into BL-21 Ecoli. Grow overnight on a plate + Kanamycin

Day 2

Transfer a colony from the plate into 20 ml LB+Kana and grow overnight

Day 3

Seed 8 Liters 2XYT +Kana with the above broth, grow to OD600 0.6-0.8

Add IPTG and grow additional 3-6 hours, harvest bacteria pellet

Day 4

Sonicate the pellet to release the protein, add ammonium sulfate to clear junk, spin

Start dialysis of cleared cell lysate with 6M Urea pH 8

Day 5

Change dialysis fluid on lysate

Day 6

Change dialysis fluid on lysate and equilibrate DE52 beads for column purification

Day 7

Initial column purification with DE52 beads-takes 6 hours Save aliquots for analysis

Day 8

Run an SDS-acrylamide gel, stain with Coomassie blue to check aliquots

Start dialysis of correct aliquots with 50mM Tris solution

Day 9

Change dialysis fluid

Day 10

Change dialysis fluid, prepare the phenylsepharose column

Day 11

Final column purification with phenylsepharose beads-takes 6 hours Save aliquots

Day 12

Run an SDS-acrylamide gel, stain with Coomassie blue to check aliquots

Start dialysis of correct aliquots with dd H₂O

Day 13

Change dialysis fluid

Day 14

Change dialysis fluid

Day 15

Freeze the purified protein, place on freeze-dryer for dehydration

Day 16

Collect the dried protein, weigh, give to Charles to test and save in freezer