Preparation of 1 M Dithiothreitol (DTT) Solution

Overview

• DTT (DTT, C₄H₁₀O₂S₂, molecular weight 154.25 g/mole) is also known as Cleland’s reagent.
• DTT is commonly used for the analysis of proteins as a reducing agent.
• A high concentration of DTT (50 – 100 mM) can reduce all disulfide bonds (-S-S-) of proteins. This is an important step to analyze protein by SDS-PAGE. 
• A low concentration of DTT (1 – 10 mM) is used to stabilize enzymes and other proteins.
• DTT is highly soluble in water and forms a clear colorless solution.
• DTT is supplied as a white powder and can be purchased from several suppliers. A 1M DTT solution can be prepared by dissolving 3.86 g of DTT in water to a final volume of 25 ml.

REQUIREMENTS

Reagents and solutions
DL-Dithiothreitol (C₄H₁₀O₂S₂)
Deionized / Milli-Q water

Equipment and disposables
Falcon® screw cap graduated Conical Tubes, 50 mL (e.g., SARSTEDT #62.547.254)
Tube rotator

COMPOSITION
1 M Dithiothreitol

OBJECTIVE
Preparation of 25 ml of 1 M Dithiothreitol solution

PREPARATION

Step 1: To prepare a 25 ml aqueous solution of 1M DTT, weigh out 3.86 g of DTT (molecular weight = 154.25). Transfer it to a 50 ml screw cap graduated Tube. Add 20 ml deionized / Milli-Q water. Mix until all DTT dissolves completely.

Tip: You can dissolve DTT by rotating the tube on the tube rotator or by inverting the tube several times.

Step 2: Adjust the volume to 25 ml with deionized / Milli-Q water. Mix it again.

Step 3 (Optional): One can sterilize the solution by passing through a 0.22 μ filter unit under an aseptic condition (inside a laminar flow hood).

Caution:
Do not autoclave the solution. Autoclaving will destroy the DTT.

Storage
DTT solution should be stored at -20°C.

Tip: It is recommended to store the solutions as small aliquots.
Caution: Avoid repeated handling and multiple freeze/thaw cycles.

Applications
– Enzyme stabilization
– Enzymatic reactions
– Protein electrophoresis