Triton X114 separation

PHASE SEPARATION OF MEMBRANE AND SOLUBLE PROTEINS

This method takes advantage of the ability of aqueous solution of the

non-ionic detergent Triton x-l 14 to separate into two phase at 25-30 °C,

due to its low cloud point. Hydrophilic, soluble proteins tend to partition

into the upper aqueous phase, while hydrophobic, membrane proteins

tend to partition into the lower detergent phase.

Before use hydrophilic contaminants of the commercially triton x-l 14

must be removed following this protocol:

add a trace of bromophenol to triton x-l 14 to stain the detergent phase

wLtrm the solution to 37 °C and centrifuge

discatt upper phase and redissolve the detergent-enriched phase

repeat thhree times

Store the detergent-enriched phase as a stock at -20°C

PROTOCOL:

50 µl cell lysate (in Triton X114 4°C) or microsomes

50 µl lysis solution

LYSIS SOLUTION:

+ 45 µl lysis buffer (150 mM NaCI, 10 mM TRIS pH:7.4)

+ 1µl PMSF

+ 2 µl IAA

+ 2 µl triton X-l 14 (1% final)

Incubate 5 min / ice and and 3 min /37°C itriton x-l 14 (tritonx-100 forms a phase containing 12% detergent)

Centrifuge 10000 rpm/RT/ 2 min and collect the upper phase (soluble) into

another Eppendorf and add 25 µ1 of lysis solution to both tubes.

Icubate 5 min / ice and 3 min / 37°C

Spin 10000 rpm/RT/ 2 min and collect the upper phase of each Eppendorf and

pool the upper and the lower phase (detergent)

Repeat the incubation (ice and 37°C) another time (total: 3 cycles)