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 24ºC 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).
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 @ 4ºC.
Keep the pellet this is the cytoskelal components, CSK.
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)
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 30ºC
spin 3 min at 3000rpm at room temperature (24ºC)
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)
Spin supernatant fractions 1hr at 30psi in the airfuge.
Keep pellet, this is the membrane skeletal fraction: MSK
Keep aqueous phase (sup)
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
vortex
spin 15 min/4ºC in ufuge
discard sup
keep pellet this is the aqueous phase (Aq.)
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 @ 24º0C, 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 @ 4º0C
wash pellet with ethanol/ether (vol:vol/50:50) as in step 7 above.
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