Preparation of 1 M Dithiothreitol (DTT) Solution


  • 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.


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

1 M Dithiothreitol

Preparation of 25 ml of 1 M Dithiothreitol solution


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).

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

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.

– Enzyme stabilization
– Enzymatic reactions
– Protein electrophoresis

Follow the table to prepare DTT solutions of different concentrations and volumes.
Conc. / Volume5 ml10 ml25 ml50 ml100 ml200 ml
10 mM0.0077 g0.015 g0.0386 g0.077 g0.154 g0.309 g
100 mM0.077 g0.154 g0.386 g0.77 g1.54 g3.085 g
0.5 M0.385 g0.77 g1.93 g3.86 g7.71 g15.43 g
1 M0.77 g1.54 g3.86 g7.71 g15.43 g30.85 g

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