EMSA Probe STAT5 - 10μM

Cat. No.: E745620

등급 & 순도 10μM

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

개요

Aladdin's EMSA Probe – STAT5 is a STAT5 consensus oligonucleotide designed for EMSA . This double-stranded oligonucleotide contains a well-recognized binding site for STAT5 transcription factors, and can be used as a probe for EMSA. The nucleotide sequence of STAT5 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.

사양

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

Only product data may be presented here.

• Tested applications (e.g., EMSA workflow steps), recommended dilutions/concentrations, annealing instructions, and positive controls: Not specified for this item; refer to CoA/Spec Sheet.

Biological Roles

Scope

• This section summarizes general biology of STAT5 binding measured by EMSA; it does not describe item-specific properties.

General background (literature)

• STAT5 (Signal Transducer and Activator of Transcription 5) encompasses STAT5A and STAT5B, latent cytoplasmic transcription factors activated by cytokine and growth factor signaling via JAK kinases. Upon phosphorylation, STAT5 dimerizes, translocates to the nucleus, and binds GAS-like DNA motifs to regulate gene transcription.
• Binding motifs: canonical sequences are summarized as TTC(N)3GAA with contextual flanking bases influencing affinity. EMSA probes containing this motif enable direct visualization of STAT5–DNA complex formation under native conditions.
• Functional context: STAT5 regulates genes involved in cell cycle control, differentiation, metabolism, and immune signaling. Binding affinity and complex composition may be modulated by co-factors, chromatin context in vivo, and post-translational modifications.
• Assay significance: EMSA readouts provide a biochemical measure of sequence-specific DNA binding independent of chromatin, complementing ChIP-based occupancy measurements. Probe design and buffer composition critically determine specificity, enabling discrimination between STAT family members via motif variants or supershift antibodies.

Note

• The biological roles above are for scientific context only. This product is for research use only and is not intended for diagnostic or therapeutic applications.

Buffer Applications

Item-specific buffer recommendations: Not specified for this item; refer to CoA/Spec Sheet.

General EMSA buffer systems (literature; not item-specific)

• Binding buffer composition (typical starting point):
  • 10 mM Tris-HCl or HEPES, pH 7.5–8.0
  • 50–100 mM KCl or NaCl (optimize for specificity)
  • 1 mM DTT or TCEP (maintain protein redox state)
  • 5–10% glycerol (improves band sharpness and sample loading)
  • 0.05–0.1% NP-40 or Tween-20 only if needed to reduce nonspecific sticking (may disrupt some interactions)
  • 50–200 ng/µL poly dI·dC or salmon sperm DNA as nonspecific competitor
  • 0.1 mM EDTA unless divalent cations are required by the complex
• Electrophoresis buffers:
  • 0.5×–1× TBE for native PAGE is common; lower ionic strength can improve resolution of specific complexes. Maintain gel temperature (4–10°C) to preserve complexes.
• Probe preparation buffers:
  • Reconstitute oligos in nuclease-free water or 1× TE; anneal complementary strands in low-salt buffer then dilute into binding buffer.

Practical notes

• pH control and salt titration are key determinants of STAT5 binding specificity. Include parallel reactions lacking competitor/mutant controls to benchmark buffer performance.

Green Alternatives

Relevance

• EMSA probes are nucleic acid reagents; green considerations center on labeling chemistry, detection reagents, and buffer systems rather than organic solvent replacement.

Greener options and practices (general; not item-specific)

• Detection choices:
  • Prefer non-radioactive detection (biotin–streptavidin chemiluminescence or fluorescent dyes) over 32P radiolabeling to eliminate radiological waste and regulatory burden.
  • If using fluorophores, select robust, photostable dyes that minimize repeated exposures and re-synthesis; avoid dyes requiring hazardous activators.
• Buffers and additives:
  • Use HEPES or Tris buffers prepared with RO/DI water; avoid heavy-metal-containing buffers; minimize use of azide unless necessary for antimicrobial preservation.
  • Choose glycerol over high-concentration organic cosolvents; avoid chlorinated solvents for any cleanup steps.
• Consumables and process:
  • Scale assays to reduce waste; employ reusable casting cassettes for native PAGE; select low-toxicity gel stains instead of ethidium bromide when compatible with your detection method.
  • Implement cold-chain consolidation to reduce dry ice/ice pack usage.

Trade-offs

• Non-radioactive systems can have slightly lower sensitivity than 32P but modern chemiluminescent and near-IR fluorescent methods often achieve comparable performance with superior safety and waste profiles.

Pharmaceutical Uses

This product is an EMSA probe intended for laboratory research workflows and is not formulated or specified for pharmaceutical manufacturing or as an excipient. No pharmacopeial status, GMP grade, or clinical-use information is provided for this item.

General context (not item-specific)

• Oligonucleotide probes are commonly restricted to analytical and research applications (e.g., transcription factor binding studies). Any use in regulated environments would require dedicated GMP synthesis, full characterization, and validated specifications not provided here.

Research Use Note (from Product Data)

• For research use only.

Physical Properties

Item-specific values

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Exact MW, formula, extinction coefficient, purity, and label type: Not specified for this item; refer to CoA/Spec Sheet.

General/literature properties for oligonucleotide EMSA probes (context only)

• Physical state: typically supplied lyophilized or in aqueous buffer as a clear solution, depending on configuration and labeling.
• Solubility: highly water-soluble in nuclease-free water or low-salt TE buffers (e.g., 10 mM Tris, 0.1–1 mM EDTA, pH ~7.5–8.0). Practically insoluble in most nonpolar organic solvents.
• pKa/logP: not applicable/meaningful for polyanionic nucleic acids under physiological conditions; behavior dominated by the phosphate backbone.
• UV absorbance (literature): DNA oligos exhibit strong absorbance at 260 nm; A260 can be used to estimate concentration using an extinction coefficient computed from sequence. Exact ε for this item is not specified.
• Thermal behavior (literature): duplex melting temperature (Tm) depends strongly on length, GC content, salt, and modifications; EMSA probes are often designed with Tm well above assay temperature to ensure stable duplexing during binding.
• Stability: generally stable when stored frozen and protected from nucleases, light (if labeled), and repeated freeze–thaw. Sensitivity to depurination at low pH and to nucleases at ambient conditions is well documented.

Note: Do not treat the above as specifications; consult the product’s CoA/Spec Sheet for item-specific values.

Quality and Grades

Item-specific grade/purity details

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

How to interpret typical grades for EMSA probes (general guidance)

• HPLC-purified oligonucleotides: commonly used for EMSA to remove truncated species that can elevate background or alter mobility. PAGE purification offers even tighter length control for short probes.
• Labeling quality: for labeled probes, percent labeling and residual free label are critical. Specifications often include labeling efficiency and residual dye/biotin content. Not specified for this item.
• Annealing quality: EMSA probes are usually supplied as complementary strands that must be annealed to form duplexes; some products ship pre-annealed. Verification can be done by native PAGE.
• UV background (for chemiluminescent/fluorescent readouts): low-UV-absorbing buffers and high chemical purity improve detection S/N ratios.
• Documentation: CoA typically provides sequence (or masked sequence), OD260 units, nmol amount, extinction coefficient, purification method, labeling details, and recommended storage. For this SKU, consult the CoA/Spec Sheet for definitive quality attributes.

Practical note

• If grade is unspecified, treat the probe as research-grade. For quantitative EMSA or publication-quality figures, consider verifying duplex formation, concentration by A260, and performance with a known positive control extract.

Reaction and Applications

Manufacturer Applications (verbatim): Not specified.

Expanded context for EMSA Probe STAT5 (general literature; not item-specific)

• Core application: Electrophoretic Mobility Shift Assay to detect STAT5 binding to a cognate GAS motif. STAT5A/B dimers formed upon upstream signaling (e.g., JAK phosphorylation) bind sequence-specifically, retarding probe mobility on native PAGE.
• Use cases: compare nuclear extracts from stimulated vs unstimulated cells; evaluate binding specificity with cold competitor or mutated probes; perform supershift assays using anti-STAT5 antibodies to confirm complex identity.
• Detection modalities: radiolabeled (32P) probes, chemiluminescent biotin–streptavidin systems, or fluorescently labeled oligos are commonly employed. The label type for this item is not specified.
• Controls: include excess unlabeled specific competitor (50–200×) to ablate specific complexes, non-specific competitor to gauge background, and a mutated probe to assess motif dependence.
• Practical tips: ensure high duplex integrity by proper annealing; titrate protein to remain in the linear response range; maintain low ionic strength to favor specific binding while preventing nonspecific aggregation; incorporate glycerol (5–10%) to sharpen bands; avoid EDTA in final binding if divalent cations are required by auxiliary factors.
• Downstream: quantitative densitometry of shifted bands; excision and mass spec of complexes when compatible; parallel reporter assays to correlate binding with transcriptional output.

Reaction Conditions

Item-specific, validated conditions are not provided for this SKU; consult the CoA/Spec Sheet. The following are general EMSA conditions from the literature and should be optimized per system.

Typical EMSA binding setup (general guidance)

• Temperature/time: 15–30 minutes at room temperature (20–25°C) or 4–10°C for labile complexes; some systems benefit from 30–45 minutes.
• Probe concentration: low pM to low nM duplex probe; keep at or below Kd for best specificity. Exact concentration depends on detection method and protein abundance.
• Protein amount: titrate nuclear extract (e.g., 2–10 µg) or purified STAT5 to obtain a single, specific shifted band with minimal smear.
• Buffer: 10 mM Tris/HEPES pH 7.5–8.0, 50–100 mM KCl/NaCl, 1 mM DTT, 5–10% glycerol, 50–200 ng/µL poly dI·dC.
• Competitors/controls: include 50–200× unlabeled specific competitor, non-specific competitor, and mutated probe control. For supershift, preincubate with anti-STAT5 antibody (isotype and amount optimized empirically).

Electrophoresis (general)

• Gel: 4–6% native polyacrylamide in 0.5× TBE; prerun 15–30 minutes to stabilize temperature/ionic strength. Run cold (4–10°C) to preserve complexes.
• Voltage/time: 8–12 V/cm, 45–90 minutes depending on gel dimensions and desired separation.

Detection

• Depends on label type (radioactive, chemiluminescent, fluorescent). The specific label for this item is not specified; follow the appropriate detection kit protocol.

Safety and Handling

Item-specific hazard data

• GHS classification, signal word, pictograms, H-statements: Not specified for this item; refer to SDS.

General safety guidance for nucleic acid probes (context; defer to SDS)

• Expected hazards: Synthetic DNA oligonucleotides without toxic labels are typically considered low acute toxicity. However, handle all research reagents with appropriate laboratory precautions.
• Primary risks: contamination with DNases/RNases compromising experimental outcomes; potential irritancy of accompanying buffers; reporter chemistries (if present) may introduce specific hazards (e.g., azides, haptens, or organic dyes). Exact labeling for this item is not specified—consult SDS/CoA.
• PPE: lab coat, safety glasses, and disposable nitrile gloves. Use nuclease-free technique to prevent degradation (dedicated tips/tubes, clean surfaces, avoid touching tube rims, change gloves frequently).
• Handling: work on ice or at 4–8°C for extended bench operations. Protect from light if a photo-labile label may be present (unknown for this item). Avoid microbial contamination.
• Incompatibilities: avoid strong acids/bases (depurination/deamination), oxidants for dye-labeled probes, and nucleases. Do not autoclave solutions containing fluorophores or biotin-streptavidin complexes.
• First aid (overview): in case of eye/skin contact, rinse with water; if inhaled as aerosol or ingested, seek medical attention per institutional policy. Provide SDS to healthcare professionals.

Always consult the product SDS for authoritative safety information and emergency measures.

Solvent Selection

Applicability to this product

• Item-specific solvent recommendations are not provided; refer to CoA/Spec Sheet.

General guidance for EMSA probes (literature/context)

• Primary solvent: nuclease-free water or low-ionic-strength TE buffer (e.g., 10 mM Tris-HCl, pH 7.5–8.0, 0.1–1 mM EDTA). These maintain oligo integrity and chelate trace metals that can catalyze degradation.
• Polarity/miscibility: polyanionic nucleic acids are fully miscible with water, not with nonpolar organic solvents. Avoid alcohol-rich environments that can precipitate DNA (e.g., ≥70% ethanol or isopropanol) unless intentionally precipitating.
• Additives: for EMSA binding mixes, salts (KCl/NaCl), DTT, glycerol, and nonspecific competitor DNA (poly dI·dC) are common; choose based on protein stability. Avoid detergents that can disrupt protein–DNA interactions unless validated.
• When to choose water vs TE: use pure nuclease-free water for short-term stocks used promptly in protein-binding mixes; use TE for longer-term storage to buffer pH and chelate divalent cations; filter-sterile and DEPC-free (do not use DEPC if amines or sensitive dyes are present).
• Light and oxidation: if a fluorescent or chemiluminescent label is present (not specified for this item), avoid oxidative buffers and protect from light to minimize photobleaching.

Storage and Reconstitution

Item-specific storage and shipping (from Product Data)

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

General reconstitution and handling guidance for oligo probes (not item-specific)

• Reconstitution solvent: nuclease-free water or 1× TE (10 mM Tris-HCl, 0.1–1 mM EDTA, pH 7.5–8.0). For labeled probes, protect from light during handling.
• Concentration: prepare a convenient stock (e.g., 10–100 µM depending on assay sensitivity). Determine concentration by A260 using the extinction coefficient provided on the CoA (not specified here) or vendor calculator.
• Annealing (if supplied as single strands): mix equimolar complementary strands in low-salt buffer, heat to 90–95°C for 2–5 min, then slowly cool to room temperature or 4°C over 30–60 min. Verify duplex by native PAGE if critical.
• Aliquoting: dispense into single-use aliquots to avoid freeze–thaw cycles; store working aliquots at -20°C (as specified) and long-term stocks at -20 to -80°C as institutional practice permits. Avoid repeated thawing; keep on ice during setup.
• Stability considerations: maintain nuclease-free technique; avoid acidic conditions and strong oxidants; for fluorescent/chemiluminescent labels (unspecified), minimize light exposure.

Always defer to the item’s CoA/Spec Sheet for any specific reconstitution volumes, buffer composition, and concentration guidelines.

Structure and Identity

Item-specific identifiers

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

General/literature description (for context; not item-specific)

• EMSA Probe STAT5 denotes a short, synthetic nucleic acid probe designed for Electrophoretic Mobility Shift Assay (EMSA) to report sequence-specific binding of STAT5 transcription factors.
• Typical structural features: double-stranded DNA oligonucleotide containing a STAT5 “GAS” motif (consensus often summarized as TTC(N)3GAA) flanked by stabilizing bases to improve binding and gel resolution. Probes may be unlabeled or carry a reporter (e.g., biotin, digoxigenin, or a fluorophore) at a 5′ end, 3′ end, or internally; exact configuration for this item is not specified.
• 2D structure (conceptual): a short duplex with Watson–Crick base-paired stem; any terminal modification (if present) is appended via a linker to the phosphate backbone at a terminus. The STAT5 core motif sits centrally to minimize end-fraying effects during protein binding.
• Stereochemistry: canonical D-ribose-derived deoxynucleotides with natural base pairing if DNA; no chiral centers beyond the standard nucleic acid sugar moieties unless specialized chemistries (e.g., LNA, phosphorothioate) are used—none are specified for this item.

Synthetic Utility

Not typically applicable.

Rationale

• EMSA Probe STAT5 is a functional analytical reagent (nucleic acid probe) rather than a small-molecule building block. It is not generally employed as a substrate, reagent, or intermediate in chemical synthesis or in standard organic transformations.

Where it is relevant

• Its “utility” lies in biochemical binding assays, not in retrosynthetic planning or named organic reactions. Users seeking chemical synthesis guidance should refer instead to sections focused on assay design, buffer selection, and reaction conditions for protein–DNA interactions.

Target Specificity

Only product data may be presented here.

• Target/epitope, sequence, species reactivity, and any validation details: Not specified for this item; refer to CoA/Spec Sheet.