- 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
Deionized / Milli-Q water
Equipment and disposables
Falcon® screw cap graduated Conical Tubes, 50 mL (e.g., SARSTEDT #62.547.254)
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. / Volume||5 ml||10 ml||25 ml||50 ml||100 ml||200 ml|
|10 mM||0.0077 g||0.015 g||0.0386 g||0.077 g||0.154 g||0.309 g|
|100 mM||0.077 g||0.154 g||0.386 g||0.77 g||1.54 g||3.085 g|
|0.5 M||0.385 g||0.77 g||1.93 g||3.86 g||7.71 g||15.43 g|
|1 M||0.77 g||1.54 g||3.86 g||7.71 g||15.43 g||30.85 g|