Determination of protein solubility

To analyse the cytoplasmic solubility of CAT mutant proteins, cell pellets from cultures grown overnight in the presence of IPTG, were resuspended in 50 mM Tris.HCl (pH 8.0), 10 % (w/v) sucrose, and mixed with 50 mg/ml lysozyme in 25 mM Tris.HCl (pH 8.0), 25 mM EDTA to a final lysozyme concentration of 0.2 mg/ml. After 15 minutes of incubation at 30°C, cells were lysed by three freeze-thaw cycles and sonication (three bursts of 15 seconds with the microtip of a Vibra-Cell™ model VC500 sonicator (Sonics & Materials Inc., Dannbury, CT) set at maximally allowed power output) Suspensions were kept on ice during sonication. Complete cell lysis was checked by light microscopy. Part of this suspension was kept as sample containing total cell proteins. The rest of the lysate was centrifuged for 10 minutes at 15,000.g, and the supernatant saved as soluble fraction. The pellet containing the insoluble protein fraction was washed, and resuspended in 50 mM Tris.HCl (pH 8.0), 10 % (w/v) sucrose. SDS-PAGE analysis on equivalent amounts of total, soluble and insoluble fractions of cell proteins allowed us to estimate the percentage of CAT produced in a soluble conformation.

Bijvoorbeeld:

  1. Celcultuur (3ml) afdraaien gedurende 10 min bij 3000rpm
  2. Supernatans afnemen
  3. Pellet oplossen in 2ml A-trit (10% sucrose, 50mM Tris pH 8.0)

    4. + 8µl lysozyme-oplossing (25 mg/ml, opgelost in A-trit)

  4. 15 min op 30°C
  5. 3x vriesdooien (-70°C en kamertemperatuur)
  6. 3x 15 seconden soniceren met microtip (op ijs)
  7. 150µl afnemen

    8. + 50µl 4x laadbuffer => Totale proteïnen

  8. 150µl 10 min centrifugeren (max rpm)

    9. hiervan 120µl supernatans nemen + 40µl 4x laadbuffer

    => Oplosbare fractie

  9. Pellet wassen met 200µl MQ
  10. Pellet resuspenderen met 150µl A-trit

    11. + 50µl 4x laadbuffer =>Onoplosbare fractie

  11. Fracties opkoken gedurende 5 min
  12. 2 min afcentrifugeren

13. 20µl op SDS-PAGE

 

 

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