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Team:MIT/Protein Purification
From 2008.igem.org
TEV Purification Protocol
These volumes are for 4L LB culture split into 4 cell pellets
- Buffers:
- 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
- Collect W1-4
- Elute with 5 c.v. EB
- Collect E1-5
- Save some of each for a gel
- Collect E1-5
- 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
- collect two washes (W1, W2)
- Elute with 16mL EB and collect 4 elutions (E1-4)
- Dialyze E1 and E2 into TEV cleavage buffer overnight
- 2L –
- 5mL EDTA
- 40mL 4M NaCl
- 100ml tris
- 2L –
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 supernatant from tev cleavage and collect flow through
- Wash with 15mL BB and collect three washes
- Elute with 20mL EB and collect two elutions
