Team:TU Darmstadt/Protocols/Proteinpurification

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=== Procedure ===
=== Procedure ===
# Centrifugation of the cell suspension at 6500 rpm at 4°C for 30 min.
# Centrifugation of the cell suspension at 6500 rpm at 4°C for 30 min.
-
# Resuspend the Pellet in 100 mL [[PBS buffer]] pH 7.4  
+
# Resuspend the Pellet in 100 mL [https://2012.igem.org/Team:TU_Darmstadt/Materials/PBS PBS buffer] pH 7.4  
# Transferration of the cell suspension into the celldisrupter and squash three times at 2.7 kbar
# Transferration of the cell suspension into the celldisrupter and squash three times at 2.7 kbar
# Centrifugation at 13.000 rpm at 4°C for an hour
# Centrifugation at 13.000 rpm at 4°C for an hour
-
# Load the prepared [[IMAC column]] with the hole supernatant and collect the flowthrough
+
# Load the prepared [https://2012.igem.org/Team:TU_Darmstadt/Protocols/IMAC IMAC column] with the hole supernatant and collect the flowthrough
# Wash the column two times with 20 mM imidazole in [https://2012.igem.org/Team:TU_Darmstadt/Materials/PBS PBS buffer] pH 7.4 and collect the floqthrough
# Wash the column two times with 20 mM imidazole in [https://2012.igem.org/Team:TU_Darmstadt/Materials/PBS PBS buffer] pH 7.4 and collect the floqthrough
# Wash the column in five steps with increasing imidazole concentrations (40mM, 60mM, 100mM, 200mM and 500mM) to elute the protein and collect the flowthrough
# Wash the column in five steps with increasing imidazole concentrations (40mM, 60mM, 100mM, 200mM and 500mM) to elute the protein and collect the flowthrough

Revision as of 17:36, 16 September 2012

Proteinpurification

Materials

  • centrifuge
  • PBS buffer
  • celldisrupter
  • imidazole
  • NiCl2 solution
  • IMAC column
  • HiLoad 16/60 Superdes 75g
  • ÄKTA FPLC
  • ice

Procedure

  1. Centrifugation of the cell suspension at 6500 rpm at 4°C for 30 min.
  2. Resuspend the Pellet in 100 mL PBS buffer pH 7.4
  3. Transferration of the cell suspension into the celldisrupter and squash three times at 2.7 kbar
  4. Centrifugation at 13.000 rpm at 4°C for an hour
  5. Load the prepared IMAC column with the hole supernatant and collect the flowthrough
  6. Wash the column two times with 20 mM imidazole in PBS buffer pH 7.4 and collect the floqthrough
  7. Wash the column in five steps with increasing imidazole concentrations (40mM, 60mM, 100mM, 200mM and 500mM) to elute the protein and collect the flowthrough
  8. Load the collected flowthrough on a 12% SDS-PAGE (Schägger)
  9. Combine the collected passages of the right protein and concentrate it to an amont of 5 mL
  10. To get rid of waste and undesired proteins perform a gelfiltration with FPLC (fast protein liquid chromatography)
  11. Usage of HiLoad 16/60 Superdex 75g from GE Healthcare
  12. Equilibration of the column with PBS buffer pH 7.4
  13. Loading 5 mL of the protein solution
  14. Elute the protein with pure PBS buffer pH 7.4 and collect the samples
  15. measure the protein concentration
  16. aliquot in 1 mL stocks and freeze in liquid nitrogen
  17. store at -80°C
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