Team:MIT/Protein Purification

From 2008.igem.org

(Difference between revisions)
 
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*Dialyze E1 and E2 into TEV cleavage buffer  overnight
*Dialyze E1 and E2 into TEV cleavage buffer  overnight
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**2L – ***5mL EDTA
+
**2L –
-
***40mL 4M NaCl
+
***5mL EDTA
-
***100ml tris
+
***40mL 4M NaCl
 +
***100ml tris
Second day:
Second day:

Latest revision as of 21:08, 27 June 2008

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
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