Endotoxin (LPS) Removal from Protein Samples Using Triton X-114 Phase Separation

1. Objective

To efficiently remove endotoxins (lipopolysaccharides, LPS) from aqueous protein samples using the temperature-dependent phase separation property of Triton X-114, while retaining the target protein in the aqueous phase.

2. Principle

Triton X-114 is fully soluble in water at low temperatures (0–4°C), forming a homogeneous solution. When heated above its cloud point (~22°C), the solution separates into a dense detergent-rich phase and a lighter aqueous phase. Hydrophobic endotoxins preferentially partition into the detergent phase, while most hydrophilic proteins remain in the aqueous phase. Centrifugation accelerates phase separation, and the aqueous phase can be collected to obtain low-endotoxin protein.

Figure 1. Chemical structure of Triton X-114

3.Materials and Reagents

Protein samples containing endotoxins (e.g., recombinant proteins, antibodies)

Low-endotoxin Triton X-114 (T101474)

Low-endotoxin buffer (e.g., PBS P397924, Tris-HCl T301491), pH suitable for protein stability

Ice bath (0–4°C)

Temperature-controlled water bath (30–37°C)

Pre-cooled centrifuge

Centrifuge tubes (1.5 mL, 15 mL, etc., depending on sample volume)

Pipettes and low-binding tips

4.Equipment

Temperature-controlled centrifuge

Precision water bath or thermostatic heating block

Ice bucket

5.Pre-Experiment Preparation

Pre-cool equipment: Place centrifuge rotor and tubes at 4°C.

Prepare Triton X-114 stock solution: Dissolve low-endotoxin Triton X-114 in low-endotoxin buffer to 10–20% (w/v), clarify, and store at 4°C.

Check sample buffer compatibility: Avoid high glycerol, sucrose, strong denaturants, or other detergents; dialyze or dilute if necessary.

6.Procedure

Step 1: Add Triton X-114 and Mix

Add Triton X-114 stock solution to the protein sample. Gently mix to form a clear, homogeneous solution, ensuring Triton interacts thoroughly with endotoxins. Maintain 0–4°C and avoid bubbles.

Step 2: Cold Incubation

Incubate the mixture in an ice bath at 0–4°C for 5–15 minutes to allow Triton X-114 to interact with endotoxins. Maintain stable temperature for optimal effect.

Step 3: Warm Incubation to Induce Phase Separation

Transfer the sample to a 30–37°C water bath and incubate for 5–10 minutes until turbidity appears, forming detergent microdroplets and an aqueous phase. Accurate temperature control is essential for effective phase separation.

Step 4: Centrifugation

Centrifuge at 10,000–15,000 × g for 5–10 minutes at 30–37°C to accelerate phase separation. Typically, three layers form:Upper aqueous phase (contains target protein).

Lower detergent-rich phase (contains endotoxins).Interfacial layer (contains impurities).Maintain temperature consistent with warm incubation.

Step 5: Collect the Aqueous Phase

Carefully aspirate the upper aqueous phase into a clean, pre-cooled centrifuge tube, recovering ~90–95% of the volume. Avoid disturbing the detergent or interface layers.

Step 6: Remove Residual Triton X-114 (Optional)

If necessary, repeat steps 1–5 two to four times or use ultrafiltration, dialysis (with an appropriate MWCO membrane), or specific resin adsorption to reduce residual Triton.

Step 7: Verification

Analyze the treated protein for endotoxin levels (LAL assay), protein concentration (BCA or Bradford assay), and protein activity or structural integrity to confirm the effectiveness of the procedure.

7.Notes and Optimization Tips

8.Summary

The Triton X-114 phase separation method is an economical and effective approach for endotoxin removal from hydrophilic proteins. Key factors for success include using low-endotoxin Triton X-114, precise temperature control, careful handling of the interface, multiple cycles if necessary, and final verification of endotoxin levels.

Aladdin: https://www.aladdinsci.com/