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Team:TU Darmstadt/Protocols/Proteinpurification
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| - | == | + | == Protein purification == |
=== Materials === | === Materials === | ||
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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 [ | + | # 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 [ | + | #: continue with the protocol IF YOU HAVE an histag in your protein |
| + | # 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 | ||
Latest revision as of 21:37, 26 September 2012
Protein purification
Materials
- centrifuge
- PBS buffer
- celldisrupter
- imidazole
- NiCl2 solution
- IMAC column
- HiLoad 16/60 Superdes 75g
- ÄKTA FPLC
- ice
Procedure
- 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
- 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
- continue with the protocol IF YOU HAVE an histag in your protein
- Load the prepared IMAC column with the hole supernatant and collect the flowthrough
- Wash the column two times with 20 mM imidazole in 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
- Load the collected flowthrough on a 12% SDS-PAGE (Schägger)
- Combine the collected passages of the right protein and concentrate it to an amont of 5 mL
- To get rid of waste and undesired proteins perform a gelfiltration with FPLC (fast protein liquid chromatography)
- Usage of HiLoad 16/60 Superdex 75g from GE Healthcare
- Equilibration of the column with PBS buffer pH 7.4
- Loading 5 mL of the protein solution
- Elute the protein with pure PBS buffer pH 7.4 and collect the samples
- measure the protein concentration
- aliquot in 1 mL stocks and freeze in liquid nitrogen
- store at -80°C
