Team:Aachen/Wetlab/Protocol35

purification of proteins

creation of Lysis buffer for SDS gel (pH = 8.0):

Composition:


  1. 50 mM NaH2PO4
  2. 300 mM NaCl
  3. 10 mM Imidazol
  4. 50 mM NaH2PO4
  5. 50 mM NaH2PO4

Preparation of Samples for SDS gel from E. coli cells

  1. take 5 ml of LB-medium and inoculate with a E.coli colony
  2. incubate at 37°C over night on a shaker

  3. dilute the culture 1:100
  4. incubate the diluted culture at 37°C on a shaker

  5. as soon as an OD600 of 0.5 is hit, transfer a sample of 1000 uL into a 1.5 ml Eppendorf container and centrifuge at 13000 rpm for 1 min at RT
  6. add 40 ul 1xPBS to the sediment and resuspend
  7. add 10 ul SDS-loading-buffer and incubate on ice
  8. induce the expression of proteins by adding IPTG to the exponentially growing culture
  9. transfer the culture into a falcon and centrifuge at 4500rpm for 15 min at 4°C; discard the supernatant
  10. Bacterial pellets after centrifugation are resuspended in 2.5 ml 1x PBS each and put into a 15 ml falcon tube.
  11. add Lisozym to the cells (end concentration of 2mg/ml)and incubate for 30 minutes on ice
  12. Sonification: Amplitude: 60% and cycle: 0.5. Cells are sonificate on ice four times for 45 s each with pauses of 45 s between the different steps of the sonification.
  13. centrifuge for 20 min at 13 rpm and 4 °C.

  14. Use the supernatant for Ni-NTA-affinity chromatography.