EMSA Probe STAT3 - 10μM

Cat. No.: E745618

GRADE & PURITY 10μM

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

개요

Aladdin's EMSA Probe - STAT3 is a STAT3 consensus oligonucleotide designed for EMSA . This double-stranded oligonucleotide contains a well-recognized binding site for STAT3 transcription factors, and can be used as a probe for EMSA. The nucleotide sequence of STAT3 consensus oligo is as follows

Precautions：

Avoid heating above 40℃, as double-stranded DNA probe will be denatured at high temperatures into single strands that cannot be used in EMSA.For detailed EMSA procedures, please refer to the manual of EMSA kits from .This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.

Instructions for Use：

1.Set up the probe labeling reaction with isotopes by adding reagents in sequence as follows:Probe to be labeled (1.75µM)2µlT4 Polynucleotide Kinase 10X Buffer1µlNuclease-Free Water5µl[γ-32P]ATP (3,000Ci/mmol at 10mCi/ml)1µlT4 Polynucleotide Kinase (5–10u/µl)1µlTotal volume10µl2.To label the probe with biotin, please referred to the EMSA Probe Biotin Labeling Kit (, GS008).3.For cold competition reaction setup, the amount of unlabeled probe should be 50-100 times that of labeled probe.

Specifications

사양 및 순도

10μM

안정성 및 스토리지

Store at -20℃ for up to 1 year.

보관 조건

Store at -20°C

배송

Ice chest + Ice pads

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Product Line

생물 제품

Application Protocols

Item-specific application details

• Not specified for this item; refer to CoA/Spec Sheet for any validated protocols, probe concentration, labeling/detection method, and recommended controls.

Note

• While general EMSA workflows are described in other sections for background, only manufacturer‑validated, item‑specific protocols should be used for QC or method transfer.

Biological Roles

General biological context (literature; not an item-specific claim)

• STAT3 (Signal Transducer and Activator of Transcription 3) is a latent cytoplasmic transcription factor activated primarily by the JAK–STAT pathway. Upon cytokine/growth factor stimulation, STAT3 is phosphorylated (classically at Tyr705), dimerizes via SH2 domains, translocates to the nucleus, and binds DNA at GAS‑like motifs (consensus TTCNNNGAA).
• DNA recognition: STAT3 dimers typically engage palindromic/near‑palindromic motifs with spacing that accommodates the dimer interface. Binding affinity is influenced by flanking bases and chromatin context in vivo; EMSA probes isolate the DNA‑binding event in vitro.
• Functional scope: STAT3 regulates genes involved in proliferation, survival, inflammation, and differentiation across many cell types. Its activity is tightly controlled by upstream kinases (JAKs), phosphatases, SOCS proteins, and PIAS family inhibitors.
• Experimental utility of EMSA probes: By reporting on STAT3–DNA complex formation, EMSA probes serve as readouts of pathway activation status in cell/tissue lysates, and enable specificity tests (competition with mutated motifs; supershifts with anti‑STAT3).

Notes

• The EMSA Probe STAT3 is provided for research use only (per Product Data). No clinical or diagnostic claims are made.

Buffer Applications

Applicability

• Highly applicable: EMSA performance depends on carefully chosen binding and running buffers. Item‑specific formulations are not provided; the following are literature‑based examples.

Typical EMSA binding buffer compositions (literature examples)

• Tris‑based buffer: 10 mM Tris‑HCl pH 7.5–7.9, 50–100 mM KCl (or NaCl), 1 mM DTT, 0.5 mM EDTA, 5–10% glycerol, 0.05–0.1% NP‑40/Tween‑20, 50–100 ng/µL poly(dI·dC) as nonspecific competitor.
• HEPES alternative: 10 mM HEPES pH 7.5 with similar salt and additives; HEPES shows reduced pH temperature sensitivity compared to Tris.

Running buffer and gel (literature)

• Native PAGE in 0.5× TBE (45 mM Tris‑borate, 1 mM EDTA, pH ~8.3) using 4–6% acrylamide for protein–DNA complexes; lower percentage improves resolution of higher‑MW complexes.
• Pre‑run the gel 15–30 min to stabilize temperature/ionic strength; run at 80–120 V at 4–10 °C to maintain complex integrity.

Practical tips

• Titrate salt (50–150 mM) to balance specificity vs affinity.
• Keep DTT fresh; include BSA (e.g., 50–100 µg/mL) to reduce nonspecific adsorption if needed.
• Validate buffer by testing wild‑type vs mutant probes and performing supershifts with anti‑STAT3.

Notes

• Exact buffer recommendations for SKU E745618 are not specified; refer to the CoA/Spec Sheet if provided.

Green Alternatives

Context

• For EMSA probes, the “greenness” considerations center on detection modality, buffer constituents, and waste minimization rather than solvent choice.

Greener choices (literature/practice)

• Nonradioactive detection: prefer biotinylated or fluorescent probes with chemiluminescent or fluorescence readout over 32P‑labeled probes to eliminate radiological hazards and associated waste streams.
• Safer stains: if post‑electrophoresis DNA visualization is required, use SYBR‑family or GelRed‑type dyes (lower mutagenicity profiles than ethidium bromide; always review SDS).
• Buffer optimization: run EMSA in 0.5× TBE to reduce buffer consumption and ionic load, and reuse pre‑chilled running buffer within the same day when quality permits.
• Mini‑gels and micro‑volume reactions: scale binding reactions to 10–20 µL and use mini‑format gels to reduce reagents and waste.

Comparison table (general)

• 32P probes: highest sensitivity; radiological waste; specialized handling.
• Biotinylated probes: high sensitivity with HRP/streptavidin chemiluminescence; non‑radioactive; simple disposal.
• Fluorescent probes: direct detection; multiplexing possible; requires suitable imager; background can be higher.

Notes

• The labeling chemistry of this specific item is not specified; consult the CoA/Spec Sheet to select compatible detection workflows.

Pharmaceutical Uses

Applicability note

• Not typically applicable. EMSA probes are research reagents used to assess DNA‑binding activity of transcription factors in vitro and in cell/tissue extracts.

General context (literature; non-clinical)

• In pharmaceutical research settings, STAT3 EMSA probes may be used in target validation, pathway interrogation, and mechanism‑of‑action studies to evaluate compound effects on signaling cascades upstream of STAT3. These are exploratory laboratory applications and not part of a therapeutic product or dosage form.

Notes

• No pharmacopeial status, excipient role, or clinical formulation use is claimed or specified for this item.
• For research use only (per Product Data).

Physical Properties

Item-specific facts (from Product Data)

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.

General/literature properties for oligonucleotide EMSA probes (not item-specific)

• Physical state: typically supplied lyophilized or in aqueous buffer as a clear solution.
• Solubility: highly soluble in nuclease‑free water and common biological buffers (e.g., Tris, HEPES; literature).
• UV characteristics (literature): strong absorption at 260 nm (ε depends on base composition and length); A260 can be used to estimate concentration after reconstitution.
• pKa / logP: not applicable in the usual small‑molecule sense; behavior governed by polyanionic phosphate backbone and ionic strength of buffer.
• Duplex stability (literature): melting temperature (Tm) depends on sequence, length, GC content, and salt; many 18–30mer dsDNA probes exhibit Tm roughly in the 50–80 °C range under moderate salt (e.g., 50–150 mM monovalent cation). Exact Tm must be computed from the actual sequence.
• Storage stability (literature): oligos are stable for months at −20 °C when stored dry or in TE buffer; repeated freeze–thaw should be minimized to prevent degradation.

Notes

• No item‑specific physicochemical specifications (mass, sequence, extinction coefficient, concentration, or label) are provided here; consult the CoA/Spec Sheet for definitive values.

Quality and Grades

Item-specific facts (from Product Data)

• Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.

General considerations for EMSA probe quality (literature/practice)

• Purification level: EMSA probes are commonly synthesized as HPLC‑ or PAGE‑purified oligonucleotides to reduce n−1 sequences and enhance specific binding signals. HPLC yields high chemical purity; PAGE can yield very high length homogeneity—particularly useful for short probes.
• Labeling quality: for nonradioactive detection, high‑efficiency incorporation of biotin, DIG, or fluorophores at the 5′ (or 3′) end is important for sensitivity and low background. The presence/position of labels affects mobility; controls are advised.
• Annealing quality: for dsDNA probes prepared from two strands, stoichiometric annealing (1:1) and verification on a native gel increase assay reproducibility.
• QC metrics typically reviewed (not item‑specific): mass spectrometry trace of the oligo, analytical HPLC/PAGE profile, OD260 and calculated nmol, and, where applicable, labeling incorporation efficiency.

Notes

• Any item‑specific grade, sequence, length, and labeling information are not specified; rely on the CoA/Spec Sheet for the definitive quality attributes of SKU E745618.

Reaction and Applications

Primary application (from product name and category)

• Designed for electrophoretic mobility shift assays (EMSA) targeting STAT3 DNA‑binding activity. Item‑specific sequence/label is not specified; usage notes below are general best practices.

General/literature application details

• EMSA principle: a labeled dsDNA probe bearing a STAT3 consensus site forms a slower‑migrating complex upon binding STAT3 dimers from nuclear or whole‑cell extracts. Free probe and complex are resolved by native PAGE and detected via the probe label.
• Use cases:
  • Monitor STAT3 activation downstream of cytokines/growth factors (e.g., IL‑6 family) in cell or tissue extracts.
  • Compare binding across conditions, mutants, or inhibitors (no clinical claims implied).
  • Perform competition assays with excess unlabeled wild‑type vs mutated probes to confirm sequence‑specific binding.
  • Supershift assays with anti‑STAT3 antibodies to verify complex identity and subunit composition.
• Practical tips:
  • Keep all components on ice; assemble binding reactions last with probe added at low nM to pM levels (exact concentration depends on labeling and detection sensitivity).
  • Include nonspecific competitor DNA and optimized salt to reduce background.
  • Pre‑clear extracts if needed and quantify protein; typical nuclear protein per lane: 2–10 µg (literature).
  • Run 4–6% native polyacrylamide in 0.5× TBE at 4–10 °C to preserve complexes.

Notes

• For probe concentration, label, and exact sequence of SKU E745618, refer to CoA/Spec Sheet.

Reaction Conditions

Applicability

• Interpreted here as assay conditions for EMSA reactions. Exact conditions for SKU E745618 are not specified; the following are literature‑based guidelines to aid method development.

Typical EMSA setup (literature)

• Binding reaction (10–20 µL total):
  • Probe: low pM to low nM final, depending on detection method and instrument sensitivity.
  • Nuclear extract or purified STAT3: 2–10 µg total protein (extract) or appropriate molar ratio for purified protein.
  • Buffer: 10 mM Tris‑HCl or HEPES pH 7.5, 50–150 mM KCl/NaCl, 1 mM DTT, 0.5 mM EDTA, 5–10% glycerol, 0.05% NP‑40/Tween‑20, 50–100 ng/µL poly(dI·dC).
  • Incubation: 20–30 min at room temperature (or 4–10 °C to stabilize labile complexes).
• Competition controls: add 50–200‑fold molar excess of unlabeled wild‑type probe to compete, or mutant probe as negative control.
• Supershift: pre‑incubate extract with anti‑STAT3 antibody (e.g., 10–30 min on ice) before adding probe to generate a slower complex confirming identity.

Electrophoresis (literature)

• Gel: 4–6% native acrylamide (37.5:1), 0.5× TBE, prerun 15–30 min.
• Run: 80–120 V for 45–90 min at 4–10 °C.
• Detection: per label (chemiluminescent for biotin/DIG; fluorescence for FAM; autoradiography/phosphorimaging for 32P).

Safety and Handling

Item-specific facts (from Product Data)

• GHS Classification / Pictograms / Signal Word / H‑Statements: Not specified for this item; refer to SDS.
• Storage Conditions: Store at −20 °C.
• Shipped In: Ice chest + ice pads.

General handling guidance for oligonucleotide EMSA probes (literature/best practice)

• PPE: laboratory coat, nitrile gloves, and safety glasses are recommended when handling any laboratory reagent.
• Avoid nuclease contamination: use nuclease‑free tubes, tips, and solutions; wear gloves and change frequently to prevent RNase/DNase transfer.
• Chemical hazards: unlabeled DNA probes are generally of low chemical hazard; probes bearing labels (e.g., fluorescent dyes, biotin) are also typically low hazard. Radioactive probes (32P) require radiation safety procedures; the labeling of this item is not specified.
• First‑aid overview: in case of accidental exposure to solutions, rinse skin/eyes with water. If ingested or inhaled as aerosol/dust, move to fresh air and seek medical advice if symptoms persist. Always consult the SDS for authoritative guidance.
• Incompatibilities: avoid strong nucleases, microbial contamination, and extreme pH that can depurinate DNA (acidic) or cause strand scission (strong base). Avoid repeated freeze–thaw.

Notes

• For definitive hazard information and disposal requirements, refer to the product‑specific SDS.

Solvent Selection

Applicability note

• As a nucleic‑acid probe, this product is formulated and used in aqueous buffers rather than organic solvents. Selection focuses on buffer composition and ionic strength rather than solvent polarity in the small‑molecule sense.

General/literature guidance for buffer selection in EMSA

• Binding buffer backbone: Tris‑HCl (pH 7.4–7.9) or HEPES (pH 7.5) provide stable pH with minimal metal chelation compared to phosphate when metal‑dependent interactions are studied.
• Ionic strength: 50–150 mM KCl or NaCl commonly balances specificity vs nonspecific binding; higher salt reduces nonspecific protein–DNA interactions but may weaken specific complexes.
• Additives: 1 mM DTT (or TCEP) to maintain protein redox state; 0.1% NP‑40 or Tween‑20 to reduce sticking; 5–10% glycerol to stabilize complexes and aid gel loading; 0.5–1 mM EDTA if divalent metals are not required.
• Competitor DNA: poly(dI·dC) or salmon sperm DNA (e.g., 50–100 ng/µL in binding reaction) suppresses nonspecific binding.

Comparison (literature)

• Tris vs HEPES: HEPES offers superior buffering near neutral pH with lower temperature dependence; Tris is widely available and cost‑effective but pH shifts with temperature.
• K+ vs Na+: K+ often better mimics intracellular ionic conditions; Na+ may slightly alter protein–DNA affinity profiles; empirical optimization is recommended.

Storage and Reconstitution

Item-specific facts (from Product Data)

• Storage Conditions: Store at −20 °C.
• Shipped In: Ice chest + ice pads.

Reconstitution and handling (general/literature guidance; not item-specific)

• Reconstitution solvent: nuclease‑free water or TE buffer (10 mM Tris‑HCl, 0.1–1 mM EDTA, pH ~7.5–8.0). Final concentration typically 10–100 µM for stock solutions; adjust per detection sensitivity.
• Annealing (for dsDNA probes supplied as single strands): mix equimolar strands in annealing buffer (e.g., 10 mM Tris‑HCl, 50 mM NaCl, 1 mM EDTA), heat to 90–95 °C for 2–5 min, then cool slowly to room temperature or in a thermocycler (−1 °C/min) to form duplex.
• Aliquoting: prepare single‑use aliquots to minimize freeze–thaw. Store working aliquots at −20 °C (short‑term) and avoid repeated cycles.
• Handling: keep on ice during setup; use nuclease‑free plastics and change gloves frequently to prevent DNase contamination.

Stability (general)

• Lyophilized DNA is typically stable for months at −20 °C; aqueous stocks are stable for weeks to months at −20 °C, longer at −80 °C. Avoid multiple freeze–thaw cycles.

Notes

• Any item‑specific concentration, label, counter‑ion, or buffer composition at delivery is not specified; consult the CoA/Spec Sheet.

Structure and Identity

Item-specific facts (from Product Data)

• Product: EMSA Probe STAT3 (SKU: E745618)
• CAS: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.
• SMILES / InChI / InChIKey: Not specified for this item; refer to CoA/Spec Sheet.

General/literature description of this product class

• An “EMSA Probe STAT3” typically denotes a short, double‑stranded oligonucleotide containing a high‑affinity STAT3 response element (e.g., GAS motif consensus TTCNNNGAA; literature) used to detect STAT3–DNA complex formation by electrophoretic mobility shift assay (EMSA).
• Structural features (literature):
  • Double‑stranded DNA (dsDNA) or annealed oligos, often 18–30 nucleotides total length, with a central STAT3 binding site.
  • May be end‑labeled (e.g., biotin, DIG, fluorescein, or 32P; exact label not specified for this item) for detection after native PAGE.
  • 2D depiction (conceptual): two antiparallel nucleotide strands with Watson‑Crick base pairing; central palindromic/near‑palindromic recognition element for STAT3 dimers.

Notes

• Exact sequence, length, modifications, counter‑ions, and label (if any) are not specified for this item; refer to CoA/Spec Sheet.

Synthetic Utility

Applicability note

• Not a small‑molecule building block or synthetic intermediate. As an oligonucleotide probe, it is not employed in typical organic synthesis workflows.

Relevant laboratory utility (literature/practice)

• Affinity/interaction studies: STAT3 EMSA probes can be used to study protein–DNA interactions, compare binding of STAT3 variants, and evaluate effects of post‑translational modifications or small molecules on DNA binding.
• Pull‑down/immobilization (conceptual): when appropriately modified (e.g., 5′‑biotin; modification not specified for this item), probes can be immobilized on streptavidin matrices for qualitative capture of STAT3‑containing complexes prior to analysis. This is an application‑adjacent utility rather than chemical synthesis.

Notes

• For synthetic chemistry applications, refer to other categories (e.g., linkers, monomers). For this item, focus on EMSA and protein–DNA interaction assays.

Target Specificity

Item-specific facts (from Product Data)

• Target name: STAT3 (Signal Transducer and Activator of Transcription 3) — inferred from product name.

Not otherwise specified

• Sequence, binding motif variant, label chemistry, validated species/cell lines, and any cross‑reactivity data are not specified for this item; refer to CoA/Spec Sheet.

General/literature context

• STAT3 binds GAS‑like DNA elements with consensus TTCNNNGAA as a dimer; specificity can be confirmed by competition with mutated motifs and supershift with anti‑STAT3 antibodies in EMSA.