Team:Aachen/Wetlab/Protocol34

Ni-NTA-affinity chromatography

lysis buffer:


  1. mix:

    • 50 mM NaH2PO4
    • 300 mM NaCl
    • 10 mM imidazole

  2. adjust pH with NaOH to 8.0

washing buffer:


  1. mix:

    • 50 mM NaH2PO4
    • 300 mM NaCl
    • 20 mM imidazole

  2. adjust pH with NaOH to 8.0

elution buffer:


  1. mix:

    • 50 mM NaH2PO4
    • 300 mM NaCl
    • 250 mM imidazole

  2. adjust pH with NaOH to 8.0

chromatography:


  1. 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)

  2. the supernatant of the lysate is given onto the matrix (keep 40 ul as sample)
  3. take 40 ul of the flowthrough as sample
  4. wash 2x with 5 ml of washing buffer (take a 40 ul sample)
  5. elution of the proteins is done in three steps with 0.7 ml of elution buffer (keep a 40 ul sample of each)
  6. the samples are stored on ice

  7. 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
  8. centrifuge short at 13000 rpm/RT
  9. pipette 10 ul of the protein marker and 20 ul of each sample into the pockets of the prepared SDS page gels
  10. electrophoresis is done at 160V, 400mA for 58 min

Comassie blue for protein identification:

Procedure

  1. After SDS-PAGE, wash the gel with ddH2O.
  2. put the gel for 20 minutes in Comassies solution for coloration and shake the container (Protocol: Coomassie).
  3. 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.