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> Fractionation of Tissue Culture Cells


Nitrogen Cavitation of Tissue Culture Cells


> Phase Partitioning


> P100 Membrane Prep from Tissue Culture Cells Method 1

      > P100 Membrane Prep from Tissue Culture Cells Method 2

Phase Partitioning

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The basic principle of the detergent phase partitioning method is to solubilize the membranes (P100) in the detergent Tx114. Cytoskeletal components (CSK) of the membranes are not soluble in the detergent and are pelleted out by centrifugation. Following phase separation at 24C centrifugation can be used to separate hydrophobic membrane proteins contained in the detergent phase (DET) from the luminal contents and membrane skeleton components Finally a last centrifugation step at 150.000 x g separates the aqueous phase (Aq), which consists mainly of soluble hydrophilic proteins, and the membrane skeleton phase (MSK).

  1. To 25ul P100 in Dog Buffer C add
    1ul DTT (of 1M DTT)
    63ul 2x solubilisation buffer
    10ul PIN (of 200x PIN)
    10ul PMSF (of 100 mM PMSF)
    37ul H20
    incubate for 5 min on ice  spin 10 min in centrifuge at 6000 - 8000 rpm @ 4C.
  2. Keep the pellet this is the cytoskelal components, CSK.
  3. To further fractionate the supernatent:
      layer on 300ul sucrose cushion
      incubate 3min at 300C ( upper layer should turn cloudy)
      centrifuge 3 min at 3000 rpm in ufuge (240C)
  4. Remove supernant (100ul) and add to it:
      10ul PIN (of 200x)
      10ul of 10% Tx114 (Tx114 diluted in MQ water)
      incubate 3 min on ice (should turn clear)
      layer back on same sucrose cushion (see cushion of step 3)
      incubate 3 min at 30C
      spin 3 min at 3000rpm at room temperature (24C)
  5. Keep the bottom, detergent phase. See 8 for further processing.

    Remove supernatent (200ul) and add:

      10ul PIN
      20ul of 10% Tx114
      incubate 3 min on ice
      layer on new cushion (300ul)
      spin out detergent 3 min at 3000rpm @ 240C
      discard detergent phase (bottom) and keep supernatant (270ul)
  6. Spin supernatant fractions 1hr at 30psi in the airfuge. Keep pellet, this is the membrane skeletal fraction: MSK Keep aqueous phase (sup)
  7. Add 1/2 vol 50% TCA to supernatant and then:
      incubate 15 min on ice
      spin 15 min in the microfuge, full speed, 40C, discard supernatant
      add 500ul of ethanol/ether (50:50 vol:vol) to the pellet (this step washes out the remains of the TCA but be careful with ether it is highly flammable
      spin 15 min/4C in ufuge
      discard sup
      keep pellet this is the aqueous phase (Aq.)
  8. To the bottom detergent phase from step 4
      resuspend in 500ul wash buffer (+ 1x PIN, + 1 mM PMSF)
      incubate 3 min on ice
      layer on new cushion (600ul)
      Spin 3 min/3000rpm in ufuge @ 240C, discard supernatant
      resuspend pellet in 500ul wash buffer
      add 1/2 vol. 50% TCA
      incubate 15 min on ice
      spin 15 min full speed in ufuge @ 40C
      wash pellet with ethanol/ether (vol:vol/50:50) as in step 7 above.
      spin 15 min/ufuge/full speed @ 4C, discard supernatent.
      keep pellet this is the detergent phase: Det.
  9. Solubilize each pellet in 80ul of tricine gel loading buffer and run 20ul/well on a Tricine SDS protein gel
    Sucrose cushions 1X Solubilization Buffer Wash Buffer
    6% Sucrose 10mM Tris/HCl pH 7.4 10mM Tris/HCl pH 7.4
    10mM Tris/HCl pH 7.4 150mM NaCl 150mM NaCl
    150mM NaCl 5% Glycerol  
    0.06% Triton X114 1% Triton X114  
    1% Glycerol