Team:MIT/Protein Purification

From 2008.igem.org

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

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
Retrieved from "http://2008.igem.org/Team:MIT/Protein_Purification"