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Latest revision as of 01:15, 18 October 2018
Ni-NTA-affinity chromatography
lysis buffer:
- mix:
- 50 mM NaH2PO4
- 300 mM NaCl
- 10 mM imidazole
- adjust pH with NaOH to 8.0
washing buffer:
- mix:
- 50 mM NaH2PO4
- 300 mM NaCl
- 20 mM imidazole
- adjust pH with NaOH to 8.0
elution buffer:
- mix:
- 50 mM NaH2PO4
- 300 mM NaCl
- 250 mM imidazole
- adjust pH with NaOH to 8.0
chromatography:
- equilibrate the chromatography-matrix
- pipette 500 ul of Ni-NTA-agarose into the chromatography-column
- wash 3x with 2 ml of lysis buffer
- wash again with 3 ml of lysis buffer (make sure the matrix does not run dry)
- the supernatant of the lysate is given onto the matrix (keep 40 ul as sample)
- take 40 ul of the flowthrough as sample
- wash 2x with 5 ml of washing buffer (take a 40 ul sample)
- elution of the proteins is done in three steps with 0.7 ml of elution buffer (keep a 40 ul sample of each)
- the samples are stored on ice
- take 40 ul of each sample and mix it with 10 ul of loading buffer and heat them for 5-10 min in a waterbath
- centrifuge short at 13000 rpm/RT
- pipette 10 ul of the protein marker and 20 ul of each sample into the pockets of the prepared SDS page gels
- electrophoresis is done at 160V, 400mA for 58 min
Comassie blue for protein identification:
Procedure
- After SDS-PAGE, wash the gel with ddH2O.
- put the gel for 20 minutes in Comassies solution for coloration and shake the container (Protocol: Coomassie).
- Wash with water and then use de-colorating solution to eliminate the blue coloration of the gel. Tip: if your solution is blue, you should change the de-colorating solution for the gel.