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. It forms a clear colorless solution in water.

Requirement

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

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

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 beaker/conical flask. Add 20 ml deionized / Milli-Q water. Mix until all DTT dissolves completely.

Tip:
One can use manual shaking using a glass pipette to mix the ingredients. The magnetic stirrer makes the dissolving process easy and convenient.

Caution:
Do not dissolve in 25 ml of deionized / Milli-Q water. In most cases, solution volume increases when a large amount of solute dissolves in a solvent.

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 aseptic condition (inside 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

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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