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Team:MIT/Protein Purification
From 2008.igem.org
TEV Purification Protocol Volumes for 4L LB culture split into 4 cell pellets
Binding Buffer: 20mM tris +.5M NaCl Elution Buffer: 20mM tris +.5M NaCl + 300mM Imidazol
Re suspend pellets in 30mL BB per cell pellet into centrifuge tube Lyse cells with sonicator Sonicate for 1min Ice for 1min Repeat 4-5 times Centrifuge at 14,000 for 40miin Save supernatant and pellet for gel Filter supernatant
Prepare 2 NiNTA columns Add 5mL NiNTA to each Wash with 1 column volume ddwater Equilibrate with 4 c.v. BB Load filtered supernatant Collect FT Save some for gel Wash with 8 c.v. BB Collect W1-4 Save some of each for a gel Elute with 5 c.v. EB Collect E1-5 Save some of each for a gel Add 1mM EDTA and 1mM DTT to all elution tubes
Run a gel.
Peptide Purification Protocol
First day: Lysis buffer:
200mL 1x binding buffer 200 mg lysozyme 40 μL 5mM AEBSF 4 enzyme tablets
Thaw frozen cell pellets
Resuspend cells in 30mL lysis buffer. Sonicate for one minute to homogenize. Rock at 4C for 2 hrs to lyse Spin lysate 40min and filter
Put 4mL Ni-NTA into flow tubes, wash w/ water and equilibrate w/ 8mL BB Load filtered supernatant and collect flow through (FT) Wash with 16mL BB and collect two washes (W1, W2) Elute with 16mL EB and collect 4 elutions (E1-4)
Dialyze E1 and E2 into TEV cleavage buffer overnight 2. 2L – 5mL EDTA 40mL 4M NaCl 100ml tris
Second day: Add 300 μL tev protease to each Cleave for two hours at rm temp Spin down for 10min at 3500rpm
Add 1mL Ni-NTA resion to columns Wash with water Equilibriate with 8mL BB Load supernatnat from tev cleavage and collect flow through Wash with 15mL BB and collect three washes Elute with 20mL EB and collect two elutions
