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DTT (dithiothreitol) (Thermo Scientific™)

Thermo Scientific DTT (DL-Dithiothreitol; Clelands reagent) is used to stabilize enzymes and other proteins, which possess free sulfhydryl groups. It has been shown to restore activity lost by oxidation of these groups in vitro. DTT quantitatively reduces disulfide bonds and maintains monothiols in a reduced state (see Reference 1).

At a final 0.1 M concentration, DTT is also widely used for disruption of protein disulfide bonds in SDS-polyacrylamide gel electrophoresis.

Highlights

• Free of endo-, exodeoxyribonucleases, ribonucleases, and phosphatases.

Applications

• Enzyme stabilization
• Enzymatic reactions
• Protein electrophoresis

Note
• DTT is not stable in solution. Only freshly-made DTT solutions should be used. To prepare a 1 M DTT solution, dissolve 1.55 g of DTT powder in 10 mL of deionized water (e.g. Water, nuclease-free, #R0581).

Pierce™ Premium Grade TCEP-HCl (Thermo Scientific™)

Thermo Scientific Pierce Premium Grade TCEP-HCl is our highest quality formulation of this disulfide reducing agent, specially characterized for applications where product integrity and risk minimization are paramount.

Features of Premium Grade TCEP-HCl:

TCEP—a thiol-free, odorless yet potent reducing agent for cleaving disulfide bonds
High quality—identity and purity confirmed by several tests, including quantitative NMR
Product integrity—enhanced level of testing and characterization compared to standard grade
Lot retention—ample supply of past lots retained to ensure future process testing
Change management—Change Control Notification (CCN) service
Consistent manufacture—batch-specific manufacturing documentation review

Compared to the standard grade product, Premium Grade TCEP-HCl provides more clearly defined quality and product support by including (a) increased analytical testing and product characterization, (b) greater batch-specific information and quality assurance review, (c) extensive lot sample retention and (d) change control notification. TCEP-HCl is pure, crystalline Tris(2-carboxyethyl)phosphine hydrochloride, a thiol-free compound that is highly effective at reducing protein and peptide disulfide bonds, as well as being odorless and stable. TCEP selectively and completely reduces even the most stable water-soluble alkyl disulfides over a wide pH range.

Advantages of TCEP-HCl over other reducing agents:
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 to 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, (e.g. histidine-tagged protein purification, maleimide conjugations).

Related Products
Pierce™ TCEP-HCl

Dithiothreitol (DTT) (Invitrogen™)

Disufide crosslinks of cystines in proteins can be reduced to cysteine residues by dithiothreitol (DTT).

Pierce™ TCEP-HCl, No-Weigh™ Format (Thermo Scientific™)

Thermo Scientific Pierce TCEP-HCl is a potent, versatile, odorless, thiol-free reducing agent with broad application to protein and other research involving reduction of disulfide bonds. The unique compound is easily soluble and very stable in many aqueous solutions. TCEP reduces disulfide bonds as effectively as dithiothreitol (DTT), but unlike DTT and other thiol-containing reducing agents, TCEP does not have to be removed before certain sulfhydryl-reactive crosslinking reactions.

Thermo Scientific No-Weigh products are specialty reagents provided in a pre-aliquoted format. The pre-weighed packaging prevents the loss of reagent reactivity and contamination over time by eliminating the repetitive opening and closing of the vial. The format enables use of a fresh vial of reagent each time, eliminating the hassle of weighing small amounts of reagents and reducing concerns over reagent stability.

Features of TCEP-HCl:

Odorless—Unlike DTT or BME, TCEP is odor-free, so it can reduce proteins conveniently on the bench; contributes to a healthier lab environment
Specific—selective and complete reduction of even the most stable water-soluble disulfides over a wide pH range
Simple—effective reduction at room temperature and pH 5 in less than five minutes
Stable—it's inherent stability and resistant to air oxidation eliminates the need for any special precautions while handling or storing; non-volatile and non-reactive toward other functional groups found in proteins
Efficient—For most applications, 5 to 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, (e.g. histidine-tagged protein purification, maleimide conjugations).

Considerations for use of TCEP:
• 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 or alkaline 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.

10X Bolt™ Sample Reducing Agent (Invitrogen™)

Bolt Sample Reducing Agent (10X) is used to reduce protein samples for protein gel electrophoresis. It contains 500 mM dithiothreitol (DTT) for a 10X concentration in a stabilized liquid form.

See all available buffers and reagents available for SDS-PAGE

UltraPure™ Dithiothreitol (Invitrogen™)

UltraPure™ Dithiothreitol (DTT, Cleland's reagent), which reduces disulfides to their corresponding thiols, is used at low concentrations to stabilize enzymes containing free sulfhydryl groups. Higher concentrations of DTT are used to cleave disulfide linkages in polypeptides and to facilitate protein denaturation by detergents or chaotropic agents.

Pierce™ Immobilized TCEP Disulfide Reducing Gel (Thermo Scientific™)

Immobilized TCEP Disulfide Reducing Gel eliminates the need to use laborious and inefficient gel filtration methods to separate the reduced sample from the reducing agent. Immobilized TCEP Disulfide Reducing Gel may be adapted conveniently to a variety of scales and formats (i.e. batch, spin column and gravity-flow column procedures). This reducing gel is for single-use applications; it cannot be regenerated and reused.

Features of Immobilized TCEP Disulfide Reducing Gel:

Odorless—reduce proteins at your bench; contributes to a healthier lab environment
Stable—virtually no loss of activity when stored refrigerated
Versatile—reduces peptides and proteins over a broad range of pH, salt or detergent concentrations, and temperature conditions
Flexible—resin slurry provides flexibility for use in spin tube, column or batch modes
Time-saving—no soluble reductant removal saves time and conserves sample
Efficient—cleaves both natural disulfide bonds and those incorporated by crosslinking

Advantages over soluble disulfide reducing agents:
Excellent recovery free from reducing agent—Removing the reducing agent and recovering the reduced molecule without sample loss is a problem inherent to DTT or β-mercaptoethanol (BME). Immobilized TCEP allows you to recover reduced protein/peptide in high yield (85% or higher) without the need to dialyze or desalt.
Odorless—Unlike DTT or BME, Immobilized TCEP is odor-free, so reductions can be carried out conveniently on the bench top.
Stable in air—The inherent stability of TCEP eliminates any need for special precautions to avoid oxidation when handling, using or storing Immobilized TCEP Disulfide Reducing Gel.
Easy to handle—Immobilized TCEP reductant, allows you to easily dispense the amount of support required for each application. Reductions can be conducted over a broad range of pH values (pH 4 to 9) and temperatures (5 to 95°C).

Trialkylphosphines serve as excellent agents for the reduction of disulfide bonds in proteins, peptides and other disulfide bond-containing molecules[1,2] and are relatively unreactive toward other functional groups.[3] The trialkylphosphine TCEP is stable in aqueous solutions and does not undergo the rapid oxidation that often occurs with other reducing agents such as dithiotreitol (DTT) and β-mercaptoethanol (BME).[4] TCEP does not interfere with common sulfhydryl-reactive reagents (e.g., maleimide crosslinkers).[5] Nevertheless, many protocols require recovery of the reduced sample separate from the reducing agent. Immobilized TCEP Disulfide Reducing Gel enables sample reduction and immediate recovery of reduced sample free of reducing agent.

Considerations for use of Immobilized TCEP Disulfide Reducing Gel:
• Reduction occurs over a wide range of pH (pH 4.0-9.0) and temperature (5°-95°C).
• Most proteins are reduced efficiently without a denaturant. However, adding a denaturant such as guanidine·HCl may aid in exposing internal disulfides to the Immobilized TCEP.
• Urea is not recommended as a denaturant as it forms cyanates that react with sulfhydryl groups.
• Do not allow metals to contact the Immobilized TCEP as this will decrease its activity.
• Including 5—20 mM EDTA in the sample buffer during reduction helps prevent reoxidation of the sulfhydryl groups by divalent metals such as Zn 2+, Cu 2+ and Mg 2+.
• The reduced sample should be used immediately after reduction because disulfides will reform over time.

Related Products
Pierce™ TCEP-HCl

USB Dithiothreitol (DTT), 0.1M Solution (Thermo Scientific™)

Product Specifications
MB Grade

Form: 0.1M solution of USB Dithiothreitol (DTT) in high purity dH2O. Solution is 0.2 µm filtered and dispensed into vials.

Containment Testing:
      DNase(endo): Passes test
      DNase(exo): Passes test
      Rnase: Passes test

Bond-Breaker™ TCEP Solution, Neutral pH (Thermo Scientific™)

Thermo Scientific Bond-Breaker TCEP Solution, Neutral pH is a stable, 0.5M solution of the thiol-free, phosphine-based TCEP compound, useful as a 10X stock for addition to SDS-PAGE sample loading buffers to reduce protein disulfide bonds.

Features of Bond-Breaker TCEP Solution, Neutral pH:

Odorless—unlike DTT and β-ME, TCEP is odor-free, contributing to a healthier lab environment
Efficient—5 to 50 mM TCEP thoroughly reduces most peptide or protein disulfide bonds within a few minutes (i.e., just as effective as DTT)
Specific—selective and complete reduction of even the most stable water-soluble alkyl disulfides
Fast—reduces protein disulfides 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
Versatile—reduces peptides and proteins over a broad range of pH, salt, detergent and temperature conditions
Compatible—removal of the reducing agent is not necessary before most applications, (e.g. histidine-tagged protein purification, maleimide conjugations), because TCEP does not contain sulfhydryl groups

Bond-Breaker TCEP Solution is a potent, odorless, thiol-free reducing agent with broad application to protein and other research involving reduction of disulfide bonds. This product is an effective and convenient replacement for B-mercaptoethanol or DTT in SDS-PAGE sample buffers. The neutral pH of this reagent provides sharp bands and avoids exposing proteins to the strong acid of TCEP·HCl, which can result in hydrolysis and carbohydrate modification.

Considerations for use of Bond-Breaker TCEP Solution:
• Reduction occurs over a wide range of pH (pH 4.0-9.0) and temperature (5 to 95°C).
• Most proteins are reduced efficiently without a denaturant. However, adding a denaturant such as guanidine·HCl may aid in exposing internal disulfides to the Immobilized TCEP.
• Urea is not recommended as a denaturant as it forms cyanates that react with sulfhydryl groups.
• Do not allow metals to contact the TCEP solution as this will decrease TCEP activity.
• Including 5 to 20 mM EDTA in the sample buffer during reduction helps prevent reoxidation of the sulfhydryl groups by divalent metals such as Zn 2+, Cu 2+ and Mg 2+.
• The reduced sample should be used immediately after reduction because disulfides will reform over time.

Related Products
Pierce™ TCEP-HCl
Pierce™ DTT (Dithiothreitol)

DTT, 1M

DTT is an essential component for kinase assays using LanthaScreen® Technology. Invitrogen's DTT is 1M in water. It is tested for background fluorescence, making it optimal for use in high-throughput screening applications.

NuPAGE™ Sample Reducing Agent (10X) (Invitrogen™)

NuPAGE Sample Reducing Agent (10X) is used to reduce protein samples for protein gel electrophoresis. It contains 500 mM dithiothreitol (DTT) for a 10X concentration in a stabilized liquid form.

See all available buffers and reagents available for SDS-PAGE

Pierce™ 2-Mercaptoethanol (Thermo Scientific™)

Thermo Scientific Pierce 2-Mercaptoethanol (2-ME), also called beta-mercaptoethanol (b-ME), is a mild reducing agent for cleaving protein disulfide bonds.

Features of 2-mercaptoethanol:

• Also known as β-mercaptoethanol, beta-mercaptoethanol, bME, b-ME
• Mild but effective reducing agent, often included in enzyme solutions to protect against catalytic site inactivation due to cysteine sulfhydryl oxidation/disulfide formation; added at final concentrations of 5 and 20 mM, with or without EDTA, as an additional protectant

Related Products
Pierce™ TCEP-HCl
Pierce™ DTT (Dithiothreitol)

Pierce™ Immobilized Reductant Column, 2 mL (Thermo Scientific™)

The Thermo Scientific Pierce Immobilized Reductant Column contains beaded resin to which an thiol-based reducing agent has been immobilized to enable solid-phase reduction of peptide and protein disulfide bonds.

Features of the Immobilized Reductant Column:

• Eliminates soluble components which interfere with the assay of free thiol groups
• No need to remove reducing agent after the reaction
• Reduced protein, free of contaminating thiols, can be easily assayed using the BCA method
• Solid phase; protocols for both peptides and proteins
• Excellent protein recovery
• Regenerable matrix useable up to four times

Both the mercaptan and the DTT methods of reduction use soluble reducing agents. This requires that the reducing reagents be removed from the sample before further procedures can be performed. Measurements of free sulfhydryl content cannot be made in the presence of these agents, and they must be removed prior to crosslinking. Removal of a soluble reductant can be difficult when a peptide is involved, and extensive desalting may result in protein loss or extreme dilution when small quantities of protein are reduced.

Pierce Immobilized Reductant Columns were developed to eliminate these difficulties, and are highly effective for reducing disulfide bonds. Immobilized TCEP Disulfide Reducing Gel is also available for reduction of peptides and proteins without contaminating your sample with reducing agent.

Related Products
Pierce™ Immobilized TCEP Disulfide Reducing Gel

Pierce™ DTT (Dithiothreitol), No-Weigh™ Format (Thermo Scientific™)

Thermo Scientific Pierce DTT (Dithiothreitol) is offered as 7.7 mg x 48-tube microplates for convenient preparation of 100 µL of 0.5M stocks for addition to protein samples for effective reduction of disulfide bonds.

Dithiothreitol is a popular reducing agent for many protein research applications, including reducing polyacrylamide electrophoresis (SDS-PAGE). The No-Weigh Format consists of a 48-microtube tray containing dry, stable, single-use aliquots of the dithiothreitol (DTT). Just puncture the seal of one microtube and add 100 µL of water or buffer. In just a few seconds you will have a solution of 500 mM DTT, ready for a variety of laboratory methods. Dilute as necessary or add the appropriate volume of the 500 mM stock solution to achieve the amount of reducing agent required for your specific application.

Highlights (of the No-Weigh Format):

Saves time—just add 100 µL water to make 500 mM DTT solution in seconds
Eliminates waste—makes 100 µL at a time, just the right amount for most applications
Always fresh—ensures full reducing strength for every use; no need to store stock solutions

With the convenient No-Weigh Format, a 500 mM DTT solution can be made in less than 30 seconds. The unique packaging ensures that the reducing agent is at full strength and able to protect proteins from oxidative damage or reduce any disulfides prior to electrophoresis.

Related Products
Bond-Breaker™ TCEP Solution, Neutral pH

Pierce™ Cysteine-HCl (Thermo Scientific™)

Thermo Scientific Pierce Cysteine-HCl is purified, crystalline cysteine hydrochloride salt (MW 175.6) that is useful as a sulfhydryl standard in assay with Ellman's Reagent or as an additive in protein refolding experiments.