Team:MIT/Protein Purification

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
 * 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
 * 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

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