DTT-PBS Solution (10 mmol/L) - BioReagent

Cat. No.: D1528834

GRADE & PURITY BioReagent

Storage

Store at 2-8°C

Shipped In

Wet ice

Descripción general

This product is a ready-to-use DTT-PBS stock working solution, prepared with sterile PBS phosphate buffer as the solvent, with a final DTT concentration of 10 mmol/L. DTT (dithiothreitol) is a potent sulfhydryl-reducing agent that cleaves both intra- and inter-molecular disulfide bonds in proteins and maintains protein thiol groups in a reduced state. The PBS buffer system maintains stable pH and osmotic pressure within the solution.

Specifications

Especificaciones y pureza

BioReagent

Condiciones de almacenamiento de almacenamiento

Store at 2-8°C

Enviado en

Wet ice

Este producto requiere envío en cadena de frío. Los servicios terrestres y otros servicios económicos no están disponibles.

Grado

BioReagent

Product Line

biological_products

Application Protocols

Protocol summaries below are provided as general literature guidance. Adjust to your system and consult detailed methods.

• A. Reducing proteins for SDS‑PAGE/Western blot (literature/general):

  1. Mix sample with 4× Laemmli buffer lacking reductant. 2) Add this DTT‑PBS Solution to reach 50–100 mM DTT final in the sample buffer. 3) Heat 5–10 min at 95°C (or 20–30 min at 60°C for sensitive proteins). 4) Cool and load gel. Note: Remove or account for PBS salts if they affect your electrophoresis/transfer system.

• B. Preparing reduced cysteines for maleimide labeling (literature/general):

  1. Incubate protein at 1–10 mM DTT (this product may be used directly) for 15–60 min at RT. 2) Desalt or buffer‑exchange to remove DTT. 3) React immediately with maleimide reagent at pH 6.5–7.5.

• C. Enzyme assay stabilization (literature/general):

  1. Prepare assay buffer and include DTT at 0.5–5 mM final. 2) Pre‑equilibrate enzyme in reducing buffer for 5–15 min. 3) Proceed with substrate addition and measurement.

• D. RNA workflows (reverse transcription) (literature/general):

  1. Include DTT at 5–10 mM in RT mixes as specified by the enzyme supplier. 2) Prepare fresh; avoid repeated warming cycles.

• Item-specific tested applications, recommended dilutions, positive controls: Not specified for this item; refer to CoA/Spec Sheet.

Biological Roles

• Item context: This product is intended for in vitro research only.

• DTT (literature/general):

  • Not a natural metabolite; functions as a laboratory reductant that maintains cysteine residues in the thiol state and reduces disulfide bonds in proteins and peptides.
  • By preserving reduced cysteines, DTT can maintain the activity of enzymes containing catalytic thiols and prevent oxidative aggregation of proteins during extraction and purification.
  • At neutral to mildly basic pH, DTT’s thiolate enhances nucleophilicity, enabling rapid thiol‑disulfide exchange; at acidic pH its activity diminishes.

• PBS matrix (literature/general):

  • Isotonic, near‑physiological ionic strength, commonly used to maintain osmotic balance and pH during handling of biological macromolecules.

• Experimental implications:

  • Reducing environments can modulate protein folding, disulfide bond isomerization, and redox‑sensitive signaling assays in vitro.
  • For cell‑based systems, exogenous DTT can be cytotoxic and perturbative; applications are typically limited to lysates, purified proteins, and cell‑free systems. Verify compatibility with your experimental design.

• No clinical/therapeutic claims: Use is restricted to research applications.

Buffer Applications

This DTT‑PBS Solution (10 mM) is designed as a convenient reducing buffer for biomolecule handling.

• Typical uses (literature/general):

  • Protein extraction/lysis: Add directly to lysis buffers to achieve 1–10 mM final DTT for maintaining cysteines in the reduced form.
  • Sample prep for SDS‑PAGE/Western blot: Combine with denaturing sample buffer (e.g., Laemmli) to reach 50–100 mM DTT (many protocols); heat as required.
  • Enzyme stabilization: Include 0.5–10 mM DTT in assay buffers for thiol‑dependent enzymes; this product provides a ready 10 mM source in PBS.
  • RNase inhibition in vitro: DTT at 1–10 mM is commonly used in reverse transcription and RNA handling buffers to help inactivate RNases.

• PBS considerations (literature/general):

  • Typical PBS pH ~7.2–7.4 supports DTT activity while remaining compatible with many proteins.
  • Nonvolatile salts may interfere with mass spectrometry; perform buffer exchange if needed.

• Practical tips:

  • Prepare working solutions shortly before use to limit oxidation. Keep capped; minimize headspace.
  • If metal‑catalyzed oxidation is a concern, a small amount of chelator (e.g., EDTA) can be included in the overall workflow if compatible.

• Composition specifics for this item (pH, exact PBS formulation, presence/absence of additives): Not specified for this item; refer to CoA/Spec Sheet.

Green Alternatives

DTT is effective but has odor, air sensitivity, and limited stability in solution. Depending on application, consider alternatives with improved stability or lower hazard profiles.

• Comparison (literature/general):

| Reducing agent | Aqueous stability | Effective pH range | Pros | Cons | |---|---|---|---|---| | TCEP·HCl | Excellent (air‑stable) | ~1–8+ | Odorless, compatible with many salts; does not contain thiols | Can reduce maleimides; may interfere with phosphine‑sensitive assays | | β‑Mercaptoethanol (BME) | Moderate | ~7–9 | Inexpensive, effective | Strong odor, volatile, can be cytotoxic; less stable than TCEP | | Dithioerythritol (DTE) | Similar to DTT | ~7–9 | Sometimes slower oxidation | Similar handling to DTT | | Ascorbate | Moderate | ~6–7 | Food‑grade, greener | Nonselective; can generate radicals with metals |

• Buffer considerations:
  • TCEP often replaces DTT in desalting‑sensitive workflows and is compatible with PBS; however, it may react with some thiol‑reactive probes. Validate for your chemistry.
  • For greener waste streams, minimize volumes, prepare only as needed, and quench spent reducing solutions by controlled aeration or metal‑free oxidation before disposal per local regulations.

Selection should be guided by the downstream chemistry (e.g., maleimide conjugation often favors DTT for initial reduction, followed by removal before coupling; TCEP may be preferred when air stability is paramount).

Pharmaceutical Uses

This product is intended for research use only and is not manufactured under GMP conditions.

• General formulation context (literature/general):

  • DTT is occasionally used in development‑stage analytical and process buffers to maintain reduced states of proteins during characterization. It is generally not used in final drug products due to odor, reactivity, and stability concerns.
  • PBS is a common laboratory buffer but may be replaced with formulation‑grade, low‑endotoxin saline/phosphate systems for development and manufacturing settings.

• Excipient status:

  • No pharmacopeial/compendial status is claimed for this item. Suitability for regulated applications would require qualification and validation of raw materials and processes.

• Recommendations:

  • For any preclinical or manufacturing use, source GMP/compendial‑grade materials and conduct full risk assessments of reductant carryover and interactions with actives, excipients, and container‑closure systems.

• Item-specific statement: For research use only.

Physical Properties

• Item-specific (solution):

  • pH, ionic strength, density, refractive index, conductivity: Not specified for this item; refer to CoA/Spec Sheet.
  • Visual appearance: Not specified for this item; refer to CoA/Spec Sheet.

• General properties of the matrix (PBS; literature):

  • Typical composition (1× PBS, literature): ~137 mM NaCl, 2.7 mM KCl, 10 mM phosphate, pH ~7.2–7.4.
  • Dielectric constant (water, 25°C; literature): ~78.4, supporting dissolution of polar/ionic solutes.

• Properties of DTT (neat compound; literature values for reference only—not the solution specification):

  • Molecular weight: 154.25 g/mol
  • Melting point: ~40–42°C (literature)
  • Solubility: Freely soluble in water; also soluble in alcohols (literature)
  • pKa values: Thiols pKa1 ~9.2; pKa2 ~10.1 (literature); reduction kinetics accelerate in mildly basic pH.
  • Redox: Strong, selective thiol-based reducing agent; oxidizes to the cyclic disulfide (DTTox).

Notes for users:

• The effective reducing activity in solution depends strongly on pH and dissolved oxygen. PBS at physiological pH maintains DTT largely in thiolate/thiol equilibrium conducive to protein work; activity diminishes in acidic media.

Quality and Grades

• Item-specific:

  • Grade: BioReagent (as provided). This denotes suitability for common biochemical and molecular biology workflows where low levels of trace contaminants (metals, particulates, organics) are important, but without the UV absorbance guarantees of HPLC/LC–MS solvent grades.
  • Matrix: Pre‑formulated PBS with 10 mmol/L DTT for convenience and reproducibility.

• What BioReagent grade implies (general guidance):

  • Typically screened for common enzymatic inhibitors and particulates; low bioburden manufacturing practices. Not equivalent to GMP or clinical grades.
  • Formulated to be ready‑to‑use in research assays (protein reduction, sample prep) with minimized batch‑to‑batch variability.

• Stabilizers and additives: Not specified for this item; refer to CoA/Spec Sheet. If chelators (e.g., EDTA) or preservatives are critical to your application, verify on the current CoA.

• Acceptance testing (examples to consider in internal QC; literature/general):

  • pH conformity to target PBS range; conductivity/ionic strength.
  • Reducing capacity assay (e.g., DTNB or cystine reduction test) to confirm 10 mM functional activity.
  • Trace metal screening (Cu, Fe) that accelerate DTT oxidation.

• Documentation: For precise lot specifications (pH, conductivity, microbial limits), consult the CoA/Specification Sheet provided with the shipment.

Reaction and Applications

Primary use: Maintenance and introduction of reducing conditions in biochemical reactions, leveraging DTT’s vicinal dithiol chemistry.

• Protein chemistry (literature/general):

  • Prevents adventitious disulfide formation during purification, storage, or enzymatic assays.
  • Reduces disulfide bonds prior to denaturation/SDS‑PAGE or proteomics workflows. Typical working ranges: 1–100 mM DTT depending on objective; this product provides 10 mM directly or can be mixed to achieve target concentrations.
  • Facilitates thiol‑specific conjugation (maleimide/iodoacetamide) by generating free cysteine residues; quench or remove excess DTT before conjugation to avoid reagent consumption.

• Nucleic acids & enzymes (literature/general):

  • Stabilizes thiol‑dependent enzymes and reduces oxidative inactivation (e.g., some polymerases and reverse transcriptases protocols employ 1–10 mM DTT).

• Mechanistic note (literature):

  • DTT reduces disulfides via a two‑step thiol‑disulfide exchange, favored at pH where thiolate forms (pH > ~7.5). Intramolecular cyclization to oxidized DTT (six‑membered ring disulfide) drives the reaction.

• Practical tips:

  • Minimize air exposure; keep capped. Prepare downstream reaction mixtures shortly before use.
  • Presence of transition metals (Cu2+, Fe3+) accelerates DTT oxidation—trace chelation (e.g., EDTA) can improve longevity if compatible.
  • Remove or dilute salts if downstream MS or ion‑exchange is planned; desalting columns or spin filters are effective.

• Notable limitations (literature):

  • DTT can reduce maleimide and interfere with click/thiol chemistries if not removed prior to coupling.
  • Ineffective at very low pH and in strongly oxidizing environments.

Reaction Conditions

Guidance below reflects literature/general practice for using DTT to reduce disulfides in biomolecules. Optimize per target system.

• pH dependence:

  • Optimal activity at pH ~7.5–8.5 where thiolate formation is favored (thiol pKa values ~9–10, literature). Activity is slower near neutral and minimal at acidic pH.

• Stoichiometry and concentration:

  • Maintenance of reduced state: 0.5–10 mM DTT is typical; this product at 10 mM can often be used as‑is or diluted.
  • Complete reduction of disulfides (denaturing conditions): 20–100 mM DTT common for proteins/peptides (literature). Combine with denaturants (e.g., SDS, urea) and heat as protocol dictates.

• Temperature and time:

  • Many proteins reduce efficiently at room temperature within 10–60 min; stubborn disulfides may require 37–60°C.

• Additives:

  • EDTA (0.5–1 mM) can suppress metal‑catalyzed oxidation if compatible. Denaturants (urea/guanidine) can improve access to buried disulfides.

• Removal before conjugation/analysis:

  • Excess DTT should be removed or quenched before maleimide/iodoacetamide labeling or MS. Use desalting columns, spin filters, or dialysis.

• Expected outcomes:

  • Efficient cleavage of reversible disulfides, formation of reduced cysteine thiols, and conversion of DTT to its oxidized cyclic disulfide (literature).

Safety and Handling

• Item-specific hazard data (GHS, signal word, pictograms, H‑statements): Not specified for this item; refer to the SDS.

• General safety guidance (literature; not a substitute for the SDS):

  • Hazard nature: DTT is a strong reducing agent and may cause skin/eye/respiratory irritation. Sulfur odor possible. PBS is nonhazardous but the mixture should be handled as a laboratory chemical.
  • PPE: Wear lab coat, appropriate gloves (e.g., nitrile), and safety glasses or face shield when splashing is possible.
  • Handling: Work in a well‑ventilated area. Minimize exposure to air to reduce oxidative loss of activity. Avoid contact with oxidizers and metal salts that catalyze oxidation.
  • Incompatibilities: Strong oxidizing agents, halogens, and transition‑metal ions (Cu2+, Fe3+) that promote thiol oxidation. Avoid nitrosating agents.
  • First aid (overview):
    • Skin/eye contact: Rinse with water for several minutes; remove contaminated clothing; seek medical attention if irritation persists.
    • Inhalation: Move to fresh air; seek medical attention if symptoms occur.
    • Ingestion: Rinse mouth; do not induce vomiting; seek medical attention.
  • Spills: Absorb with inert material, collect for disposal. Rinse area with water.
  • Waste: Collect as aqueous hazardous waste per institutional/municipal regulations; segregate from oxidizers.

Always consult the official SDS for authoritative hazard classification and emergency measures.

Solvent Selection

This product is a ready‑to‑use aqueous PBS solution and is not intended as an organic solvent. Selection considerations focus on buffer compatibility with biomolecules and downstream methods.

• Polarity/miscibility (general):

  • Highly polar, water‑based buffer; fully miscible with water and other common biological buffers.
  • Not miscible with most nonpolar organic solvents.

• When to choose this product:

  • For immediate use in protein or nucleic acid workflows requiring a defined reducing environment (10 mM DTT) at physiological ionic strength.
  • When avoiding in‑lab weighing and dissolution of DTT to reduce exposure and variability.

• Alternatives (literature/general):

  • If metal‑sensitive systems are used, consider PBS containing chelators (e.g., 0.5–1 mM EDTA) to slow DTT oxidation—verify compatibility with your assay.
  • For mass spectrometry workflows, volatile buffers (e.g., ammonium bicarbonate) with DTT may be preferred to avoid nonvolatile salts.

• Small comparison (literature/general):

  • PBS + DTT: Physiological salt, stable pH ~7.2–7.4; ideal for protein handling.
  • Tris buffer + DTT: pH buffering in 7–9 range; can enhance reduction rates; amines may interfere with some conjugation chemistries.
  • HEPES + DTT: Good biological buffering near neutral pH; zwitterionic; low metal binding.

Storage and Reconstitution

• Item-specific (from Product Data):

  • Storage temperature: Store at 2–8°C.
  • Shipping: Shipped on wet ice.

• Format: Ready‑to‑use aqueous solution; no reconstitution required.

• Stability and handling (general guidance):

  • DTT is susceptible to air oxidation in solution. Keep container tightly closed, minimize headspace, and limit repeated openings. Protect from prolonged exposure to air and elevated temperature.
  • For prolonged use, consider preparing working‑volume aliquots to avoid multiple freeze–thaw or warm–cold cycles. If freezing is necessary for your workflow, validate performance post‑thaw; some users store at ≤−20°C in aliquots, but stability depends on matrix and headspace oxygen.
  • Avoid contamination; use sterile technique if sterility is required for your application (sterility of this item is not specified; refer to CoA/Spec Sheet).

• Shelf life, pH, and exact composition: Not specified for this item; refer to CoA/Spec Sheet.

• Before use: Mix gently to ensure homogeneity; inspect for precipitate or discoloration. Discard if significant discoloration or loss of reducing activity is observed (test via DTNB or functional assay, literature).

Structure and Identity

Brief description: This product is an aqueous phosphate‑buffered saline (PBS) solution containing 10 mmol/L dithiothreitol (DTT), supplied as a ready‑to‑use reducing buffer for biochemical applications.

• Item-specific (from Product Data):

  • Concentration: DTT 10 mmol/L in PBS
  • Format: Aqueous solution (buffered saline)
  • Grade: BioReagent
  • Storage: 2–8°C; shipped on wet ice
  • CAS, InChIKey, SMILES: Not specified for this item; refer to CoA/Spec Sheet.

• Literature identity (active component, DTT; for reference only):

  • Common names: Dithiothreitol (DTT), Cleland’s reagent
  • Molecular formula: C4H10O2S2 (literature)
  • Molecular weight: 154.25 g/mol (literature)
  • SMILES: C(C@@HO)C@HO (DTT; literature)
  • InChIKey: AYFVYJQAPQTCCC-ARJAWSKDSA-N (DTT; literature)
  • Structural features (DTT, literature): A 1,4‑threitol backbone bearing two primary thiols (–SH) and two secondary alcohols (–OH); the vicinal dithiol forms a stable six‑membered cyclic disulfide upon oxidation.

• 2D structure (described in words; literature): A four‑carbon chain with two adjacent stereocenters (threitol configuration); C2 and C3 each bear a hydroxyl and a methylene‑thiol substituent, giving a vicinal dithiol diol.

Synthetic Utility

While primarily a biochemical reagent, DTT can serve niche roles in synthetic and materials chemistry (literature/general):

• Reductive transformations:

  • Selective reduction of disulfides to thiols under mild, aqueous conditions.
  • Protection/deprotection strategies involving reversible disulfide formation in peptide synthesis.

• Ligand/auxiliary roles:

  • Acts as a soft‑donor ligand for certain metal surfaces; sometimes used to control nanoparticle surface chemistry by providing thiol termini.

• Selectivity considerations:

  • DTT’s di‑thiol structure can chelate soft metals; it is relatively selective for disulfide reduction compared to carbonyls or nitro groups (which typically require other reductants).

• Practical notes:

  • Operates best near neutral to mildly basic pH; activity decreases in acidic media.
  • For organic‑phase chemistry, solubility limitations and high polarity reduce utility; aqueous/bioconjugation contexts are where DTT excels.

For most synthetic reductions outside bioconjugation or peptide work, alternative reductants (e.g., NaBH4, PPh3/DIAD for Mitsunobu, or TCEP in water) are more common.

Target Specificity

Not applicable. This product is a chemical buffer/reagent and does not have antigen/epitope specificity, clone, isotype, or species reactivity. For application relevance, refer to Reaction & Applications and Application Protocols sections.