Enrichment of integral membrane proteins with Triton X-114 phase separation

If the relative concentration of the antigen of interest is less than 0.2 % of total protein, high background signals, due to unspecific binding, make it difficult to detect the specific band. An aqueous solution of the nonionic detergent Triton X-114 is homogenous at 0°C but separates in an aqueous and detergent phase above 20°C. This property can be employed to separate integral membrane proteins from soluble hydrophilic proteins and will remove background signals.

Solutions needed:.

• Triton X-114 solution: 10 mM Tris/HCl, pH 7.4; 150 mM NaCl; 2 % Triton X-114.
  
  
• Sucrose solution: 10 mM Tris/HCl, pH 7.4; 150 mM NaCl; 6 % (w/v) sucrose; 0.06 % Triton X-114.
  

The solutions can be stored at 4°C.

Procedure:

1. Dissolve protein sample which should have a concentration of 0.2-1.0 mg/ml in Triton X-114 solution to a final Triton concentration of 0.5-1 % and incubate for 1 h on ice.
   
   
2. Apply a 200 µl layer of the sample on a 300 µl sucrose cushion.
   
   
3. Incubate for 3 min at 30°C and centrifuge at 300 g for 3 min at RT .
   
   
4. Transfer aqueous phase to a fresh tube and add Triton X-114 solution until the concentration used for the first separation is reached. Keep the detergent phase on ice.
   
   
5. Repeat phase separation. The second sucrose cushion can be applied on the detergent pellet kept on ice.
   
   

The membrane proteins are in the detergent phase. To remove the detergent a chloroform/methanol precipitation can be employed:

1. Mix 100 µl sample with 400 µl methanol and centrifuged at 9000 g for 10 sec.
   
   
2. Add 200 µl of chloroform, mix and centrifuge again at 9000 g for 10 sec.
   
   
3. Add 300 µl H₂O, mix and centrifuge again at 9000 g for 1 min.
   
   
4. Remove carefully the upper aqueous phase. Do not remove the protein which is located exactly between the two phases.
   
   
5. Precipitate the protein by adding 300 µl methanol to the organic phase and spin down the protein at 9000 g for 2 min.
   
   
6. Discard the supernatant.
   

References:

Tiruppathi C, Alpers DH & Seetharam B (1986): Phase separation of rat intestinal brush border membrane proteins using Triton X-114. Analytical Biochemistry 153:330-5.

Bordier C (1981): Phase separation of integral membrane proteins in Triton X-114 solution. Journal of Biological Chemistry 256 :1604-7.