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BIOLOGICAL REAGENTS > ER / MITO Separation

> Separation of Mitochondria and Microsomes

> Preparation of Heavy and Light Membrane Fractions From MDCK Cells

Separation of Mitochondria and Microsomes

Prepare microcentrifuge tubes by adding mitochondria import retic lysate (I have been using up to 30ml) then add mitochondria and KRMs to desired concentrations, make sure to compensate with the appropriate buffer, mix well, THEN add the translation products.
eg:

  • 24ml retic lysate for mitochondria import
  • 56ml mitochondria @ 1-4  mg/mL
  • 6ml FJ-41 KRM @ 4 eq/ml
  • 2ml each translation product (Cb5,  Bcl-2, pOCTgPA)
  • incubate 60 min. @ 30°c (mixing every once in a while)
  • place on ice for 5 min.  to cool reactions.
  • make 0.5M in KAc and 50mM in EDTA (eg to the above reaction add about 12.3ml 4M KAc (pH 7.5) and 10.9ml 0.5M EDTA (pH 7.5))  mix well.
  • let sit on ice for 15 min.
  • prepare microcentrifuge tubes containing 50ml sucrose cushion ( 0.5M sucrose/ 0.5M KAc/ 50mM EDTA/ 1/3 vol. mitochondria buffer B (same as resuspension buffer)).
  • layer import reactions on cushions
  • spin 5 min.  4°C,  10,000 rpm (Hermle, 1.5 ml tube rotor)
  • transfer supernatants to airfuge tubes containing 50ml of same sucrose cushion,  spin 20 psi,  10min.
  • meanwhile remove remaining cushions from mitochondria tubes/ pellets and resuspend mitochondria pellets in 100ml  resuspension buffer, spin 5 min. highest speed in mfuge at 4°C, remove supernatant and add 15ml  2xTricLoading buffer (2xTLB) to pellets, sonicate in bate sonicator for 30 sec, add 15ml 0.4M DTT, manually resuspend with pipet, heat at 80°C for 5 min.
  • from airfuge spin:  remove and save supernatant, discard sucrose cushion, add 30ml TLB, heat 75°C for 10 min., resuspend manually
 

Materials

 
   
bulletMicrocentrifuge tubes  
bulletRetic Lysate for mitochondrial import  
bulletBuffer  
bulletMitochondria  
bulletFJ-41 KRM  
bulletTranslation products (Cb5,  Bcl-2, pOCTgPA)  
bulletIncubator  
bulletIce  
bulletKAc  
bulletEDTA  
bulletSucrose  
bulletMitochondria Buffer B (same as resuspension buffer)  
bulletHermle, 1.5 ml tube rotor  
bulletAirfuge tubes  
bulletMicrocentrifuge  
bullet2xTricLoading buffer (2xTLB)  
bulletbate sonicator  
bulletDTT  
bulletpipet