You will need to do a reduction step in order to remove the protecting group from your oligo before using it in your reaction. One option is to use Reductacryl (a solid DTT resin) to carry out the reduction. To do this, add the Reductacryl in excess to your oligo, then run the reaction through a spin column. You can add this directly to your reaction mix. If you would prefer not to use DTT, you can also use TCEP as your reducing agent. To do this, add the TCEP in 100fold excess (ie; 30mM TCEP to 300uM oligo) and let it set for a couple of hours at room temp to reduce the oligo. You do not need to worry about removing the TCEP from your oligo before using it in your reaction. Please see the specific protocols below:
The reducing protocol is more successful when done in a pH-controlled environment, therefore we would strongly recommend using TE Buffer or some other type of buffer.
Reducing Thiol Modified Oligos
- Treatment with solid-phase DTT
DTT is available immobilized on acrylamide resin (Reductacryl™, Calbiochem Inc. Cat. No. 233157). The Reductacryl™ reagent can be used in a batch technique to reduce the disulfide bonds.
- Treatment with DTT in liquid phase
The oligo can be treated with DTT or stored in DTT, which must be removed immediately before use.
- Make a solution of oligonucleotide in TE plus DTT. We use 100 uM oligo in 10 mM DTT in 1x TE.
- Pass oligo through a large bed volume Sephadex column to remove DTT. Note that small bed volume spin columns can allow trace DTT to remain with the oligo, which can interfere with subsequent coupling reactions. As an alternative, use the extraction procedure outlined below.
- Bulk Reduction
Reconstitute the oligonucleotide (up to 1 mg of oligo can be used) in 100 ul of 2% TEA (triethylamine), 50 mM DTT and allow to stand at room temperature for 10 min. Remove DTT using one of the 3 methods outlined below:
- Extraction/PPT: extract 4x using 400 ul of ethyl acetate (layers readily separate and the DTT will partition with the ethyl acetate and the DNA will partition in the aqueous phase).
- Recover oligo by acetone precipitation or gel filtration.
- Add five volumes of acetone solution (2% LiClO4) w/w in acetone) to one volume of the oligo solution in a 14 mL tube.
- Chill the resulting solution at -20°C for 15 minutes.
- Centrifuge the sample at 2500 - 5000 RPM for 10 - 5 minutes respectively.
- Remove the supernatant.
- Dry the sample under vacuum to remove trace acetone.
- To remove LiClO4 and other salts, the sample can be washed with 2-3 mL of n-butanol centrifuged again followed by removal of the butanol supernatant.
- Size exclusion or gel filtration chromatography
- Load the sample of oligonucleotide on a Sephadex G25F column that has been thoroughly washed with distilled water.
- Elute the column with water by gravity flow and collect fractions.
- Measure the UV absorbance at 260 nm. The first eluting peak at the void volume is the oligonucleotide.
- Concentrate fractions using a SpeedVac evaporator.
Any oligonucleotide that is not used immediately should be stored frozen. Over time, the oligo will oxidize and the above procedure will need to be repeated before coupling.
TCEP·HCl Tris(2-Carboxyethyl) phosphine Hydrochloride
Odorless, water-soluble reducing agent.
The ability of trialkylphosphine compounds to reduce protein disulfide bonds have been known for many years.1,2 Phosphines are stable in aqueous solution, selectively reduce disulfide bonds, and are essentially nonreactive toward other functional groups commonly present in proteins.2 Trialkylphosphines, however, were hindered by their instability in water and their disagreeable odor. These obstacles were overcome by discovery of tris(2-carboxyethyl)phosphine (TCEP).3-17
Tris[2-carboxyethyl] phosphine (TCEP) is a popular odorless reducing agent for protein applications. The uses for TCEP have recently been expanded with the introduction of a stable, neutral pH TCEP solution, Bond-Breaker TCEP Solution, that is ideal for preparing proteins for SDS-PAGE analysis. Reducing peptides and proteins without contaminating the sample with reducing agent is now possible with Immobilized TCEP Gel where TCEP has been covalently attached to an agarose support.
TCEP selectively and completely reduces even the most stable water-soluble alkyl disulfides over a wide pH range. Reductions frequently require less than 5 minutes at room temperature. TCEP is non-volatile, odorless, and unlike most other reducing agents, is resistant to air oxidation. Compared to DTT, TCEP is more stable, more effective, and able to reduce disulfide bonds at lower pHs.
Reduction of Disulfide bonds using Bondbreaker TCEP Solution.
Advantages of TCEP·HCl over traditional alternatives for reducing disulfides:
- Odorless – Unlike DTT or BME, TCEP is odor-free, so reductions can be carried out conveniently on the bench top.
- Stable in air – The inherent stability of the TCEP moiety eliminates the need for any special precautions to avoid oxidation when handling, using or storing TCEP.
- Efficient – For most applications, 5-50 mM TCEP provides sufficient molar excess to effectively reduce peptide or protein disulfide bonds within a few minutes at room temperature.
- Compatible – With TCEP, removal of the reducing agent is not necessary prior to most applications.
- Odorless – reduce proteins at your desktop; contributes to a healthier lab environment
- Specific – selective and complete reduction of even the most stable water-soluble disulfides
- Simple – effective reduction at room temperature and pH 5 in less than five minutes
- Stable – resistant to air oxidation; nonvolatile and nonreactive toward other functional groups found in proteins
|Properties of TCEP·HCI |
|Molecular Formula ||C9H16O6PCl |
|Molecular Weight ||286.65 |
|CAS Number ||51805-45-9 |
|Solubility ||> 310 g/L (1.08 M) |
|Storage Conditions ||Store at room temperature |
Considerations for use of TCEP·HCl
- TCEP is generally very soluble in aqueous buffers at nearly any pH. Therefore, working concentrations and 10X stock solutions may be readily prepared in most aqueous buffers
- TCEP is stable in aqueous, acidic, and basic solutions. When TCEP is dissolved directly in water, the resulting pH is approximately 2.5.
- TCEP is not particularly stable in phosphate buffers, especially at neutral pH. Therefore, if TCEP is to be used in PBS buffers, prepare the working solution immediately before use.
- TCEP may be used as a substitute for DTT or 2-mercaptoethanol (2-ME) in sample loading buffer for SDS-PAGE; use a final concentration of 50 mM TCEP.
- Because TCEP is charged in solution, it is not compatible for use in isoelectric focusing.