Protocols > PSII reaction center extraction for cryo-EM

Protocol

1. Prepare Sucrose gradient:
   0.35 M Sucrose
   10 mM Hepes-KOH, pH 7.5
   0.01% w/v digitonin
   Formed by freezing and thawing.
2. Wash BBY particle:
   200 µg Chl BBY washed with 10 mM Hepes-KOH, pH 7.5
   Resuspended to a concentration of 1mg/mL in 10 mM Hepes-KOH, pH 7.5
   
   
3. Solubilization started by adding same volume of the mix:
   1% w/v digitonin ans 0.4 % w/v α-dodecylmaltoside
   (Yielding final concentration is; 0.5 mg/mL Chl, 0.5% digitonin, 0.2% α-DDM)
   
   
4. Incubate on ice 30 minutes.
   
   
5. Centrifuge 12 000 x g 10 minutes, use supernatant.
   
   
6. Load on sucrose gradient an spin:
   SW41 41 000 rpm for 7 h (200 µg Chl)
   (SW41 41 000 17 h with 0.65 M gradient)
   SW60 60 000 rpm 2.5 h (100 µg Chl)