EMSA Probe NF-κB - 1.75μM

Cat. No.: E745607

등급 & 순도 1.75μM

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

개요

Aladdin's EMSA Probe - NF-κB is a NF-κB consensus oligonucleotide designed for EMSA . This double-stranded oligonucleotide contains the well-recognized binding sequence for nuclear factor-kappa B (NF-κB), and can be used as a probe for EMSA. The nucleotide sequence of NF-κB 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

1.75μM

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

Tested applications, recommended dilutions/concentrations, gel percentages, and detection workflows for this SKU are not specified in the provided Product Data. Please consult the product’s CoA/Spec Sheet for item-specific protocols.

Note: While general EMSA procedures exist, performance parameters (e.g., probe amount, incubation time, buffer composition, detection chemistry) depend strongly on the probe’s sequence, length, and labeling—details that are not included here.

Biological Roles

Context for use (general literature): The NF-κB family comprises inducible transcription factors (e.g., p65/RelA, p50/NF-κB1, c-Rel, RelB, p52/NF-κB2) that bind κB motifs in DNA to regulate genes involved in immune signaling, stress responses, and cellular homeostasis. In resting cells, NF-κB is typically sequestered in the cytoplasm by IκB proteins; upon pathway activation, IκB is degraded, allowing NF-κB nuclear translocation and DNA binding.

Role of an EMSA NF-κB probe:

• Reports DNA-binding activity: Serves as a biochemical readout of NF-κB’s capacity to bind its cognate κB motif in nuclear extracts or purified systems.
• Subunit discrimination (with supershift): Antibody-based supershift experiments can help attribute DNA-binding complexes to specific NF-κB dimers in a research setting.
• Pathway modulation studies: Enables comparative assessment of NF-κB activation under different stimuli, inhibitors, or genetic perturbations (strictly research; no clinical inference).

Assay considerations:

• Consensus versus gene-specific sites: Probes may encode a canonical consensus κB sequence or gene-promoter-derived variants. Binding affinity and complex composition can vary accordingly (literature). The specific sequence of this item is not provided.
• Chromatin context: EMSA reflects sequence-level binding in vitro; results complement, but do not replace, cellular assays (e.g., reporter assays, ChIP).

Buffer Applications

This product functions within EMSA buffer systems rather than serving as a buffer component itself. Below are general, literature-based buffer recommendations for NF-κB EMSA; item-specific recipes are not provided.

Typical EMSA binding buffer (literature example):

• 10 mM Tris–HCl, pH 7.5–8.0
• 50–150 mM NaCl or KCl (optimize for specificity)
• 1 mM DTT (maintain protein activity)
• 0.5 mM EDTA (optional; balance nuclease protection vs metal dependence)
• 2–10% glycerol (stabilize complexes and increase sample density)
• 0.05–0.1% NP-40/Tween-20 (optional to reduce nonspecific adsorption)
• Poly(dI•dC) 25–100 ng per 10–20 µL reaction (suppress nonspecific binding)

Running and gel conditions (literature):

• Native PAGE: 4–6% polyacrylamide (29:1) in 0.5× TBE, pre-run 15–30 min; load chilled samples; run at 80–150 V at 4–10°C for best resolution of complexes.
• Sample volume: 10–20 µL with final glycerol 5–10% to ensure well entry.
• Detection: Depends on probe labeling (not specified here); follow corresponding chemiluminescent or fluorescence protocols.

Notes:

• Optimize salt, glycerol, and competitor DNA empirically for your protein source. Excess salt can abolish weak complexes; insufficient salt increases background.
• If the probe arrives as single strands, anneal in 10 mM Tris, 50 mM NaCl, pH ~7.5–8.0, heat to 90–95°C for 2–5 min, then slow-cool to room temperature before use (general practice; item format not specified).

Green Alternatives

Although this is a nucleic acid probe rather than an organic solvent or reagent, greener practice considerations apply to EMSA workflows.

Greener choices (general/literature):

• Non-radioactive labels: Prefer biotin- or fluorophore-labeled probes over 32P radiolabeling to eliminate radionuclide handling and long-lived waste.
• Aqueous-only systems: Perform all steps in water-based buffers (Tris/borate/EDTA), avoiding organic solvents that add hazards and VOC emissions.
• Energy efficiency: Run native gels at room temperature or 4–10°C as required; avoid excessive cooling unless necessary for resolution.
• Waste minimization: Scale reaction volumes (e.g., 10–20 µL) and gel sizes to experimental needs; reuse gel running buffers when compatible with data quality.

Tradeoffs (balanced view):

| Option | Advantages | Considerations | |---|---|---| | Non-radioactive probes | Safer handling; no isotope decay; chemilum/fluorescent detection | Typically lower sensitivity than 32P; requires optimized detection chemistry | | 32P-labeled probes | Very high sensitivity and linearity | Radiation safety, specialized disposal, short half-life logistics | | TBE buffer | High buffering capacity; sharp EMSA bands | Borate can interact with cis-diols (rarely relevant); proper disposal needed |

Note: This item’s specific labeling chemistry, if any, is not specified. Choose the detection route consistent with your lab’s EHS policies and sensitivity requirements.

Pharmaceutical Uses

This product is a research-grade EMSA probe. It is not intended for pharmaceutical formulation, diagnostic, or therapeutic use.

• Excipient/formulation role: Not applicable.
• Pharmacopeia status (USP/EP/JP): Not applicable for this research reagent.
• Manufacturing use: In an R&D context, such probes can support process or mechanism-of-action studies related to transcription factor–DNA interactions, but they are not used as drug substances or excipients.

Any item-specific regulatory status, GMP compliance, or QC attributes suitable for regulated environments are not specified and should be confirmed via the CoA/Spec Sheet and your quality team if needed.

Physical Properties

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Purity/Grade: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.
• Solubility: Generally water-soluble (literature, for DNA oligonucleotides), but exact solvent and concentration guidance are not specified for this item.
• pKa / logP / refractive index / BP / MP / density: Not applicable or not typically defined for oligonucleotide probes; not specified for this item.

General/literature notes for DNA probes:

• UV absorbance: Duplex/ssDNA exhibits a strong absorption band at ~260 nm (literature). The extinction coefficient depends on sequence and length (nearest-neighbor calculations); item-specific values are not provided.
• Physical form: EMSA probes are commonly supplied lyophilized or in aqueous buffer (e.g., TE, water) at defined concentration; the exact form for this SKU is not specified.
• Stability considerations: Oligonucleotides are stable when stored frozen and protected from nucleases and repeated freeze–thaw cycles (literature). Actual stabilizers (if any) and shelf-life for this item are not specified.

Bottom line:

• For accurate handling (e.g., molar calculations, A260-to-concentration conversion), the sequence, length, and extinction coefficient must be taken from the item’s CoA/Spec Sheet, which are not included in this summary.

Quality and Grades

• Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.
• Quality documentation: CoA/Spec Sheet should define sequence, length, labeling chemistry (if any), synthesis method, and post-synthesis purification (e.g., HPLC, PAGE), none of which are specified here.

Interpretive guidance (general for EMSA DNA probes):

• Purity considerations: EMSA performance benefits from high-purity, full-length duplex probes. PAGE- or HPLC-purified oligos can reduce background and smearing in gels (literature). If labeled, certification of label incorporation and residual free label content is important.
• Annealing quality: For duplex probes supplied as single strands, correct stoichiometry and annealing conditions are critical; some products are shipped pre-annealed. This item does not specify format.
• Performance metrics: Useful metrics include mass verification (MS), OD260-based quantitation, and functional testing in EMSA with a known NF-κB positive control extract—details not provided here.

What to look for on the item’s CoA/Spec Sheet:

• Sequence and motif placement; strand length(s); labeling and linker details; purification method; concentration (if solution); residual salts; recommended binding and running buffer conditions. Since these are not provided in this listing, please consult the product documentation.

Reaction and Applications

This product is designed for Electrophoretic Mobility Shift Assays (EMSA) targeting NF-κB–DNA interactions.

Core applications (general/literature):

• DNA–protein complex detection: Demonstrate NF-κB binding to a cognate κB motif via mobility retardation in native polyacrylamide gels.
• Specificity controls: Include excess unlabeled “cold” competitor probe (specific vs mutated) to validate sequence-specific binding; loss of shifted band with specific competitor indicates target engagement.
• Supershift assays: Addition of anti–NF-κB antibodies can further retard the complex (supershift), identifying subunits involved (e.g., p50, p65), provided antibodies are validated for EMSA.
• Kinetic/affinity insights: Titration of protein or probe enables estimation of apparent binding affinities and stoichiometry under the chosen buffer conditions.
• Pathway studies: Compare nuclear extracts from stimulated vs control cells to monitor NF-κB activation state via induced nuclear translocation and DNA-binding activity (no clinical claims; research context only).

Practical tips (literature/practice-based):

• Use fresh nuclear extracts, keep reactions on ice until incubation, and include protease inhibitors and DTT to preserve activity.
• Incorporate poly(dI•dC) or salmon sperm DNA to suppress nonspecific binding.
• Maintain low probe concentrations (typically low pM–nM) to reduce background; optimize salt (50–150 mM) and Mg2+ only if required.
• Run native 4–6% polyacrylamide gels in 0.5× TBE at 4–10°C for sharper resolution. For non-radioactive detection (e.g., biotin/fluor), follow the appropriate post-electrophoresis detection workflow.

Item-specific application notes are not provided in this listing; defer to the CoA/Spec Sheet for probe format and recommended conditions.

Reaction Conditions

No chemical reaction conditions are associated with this product. However, EMSA assay conditions (general/literature) determine the “binding reaction” outcomes and are outlined here for convenience. Item-specific recommendations are not provided.

Typical EMSA incubation parameters (literature):

• Temperature: 20–25°C for 15–30 min; some complexes benefit from 4°C incubation to enhance stability.
• Reaction volume: 10–20 µL.
• Protein amount: 1–10 µg nuclear extract or 0.1–1 µg purified protein per reaction (optimize empirically).
• Probe concentration: Low pM to low nM; maintain probe as the limiting reagent to emphasize specific complexes.
• Salt: 50–150 mM monovalent cation (Na+ or K+); adjust to balance specificity vs sensitivity.
• Additives: 1 mM DTT; 2–10% glycerol; 25–100 ng poly(dI•dC) per reaction; 0.05% NP-40/Tween-20 optional.
• Competition assays: 50–200× molar excess of unlabeled specific competitor; use equal amount of mutant competitor to demonstrate specificity.

Electrophoresis:

• Gel: 4–6% native PAGE, 0.5× TBE, pre-run 15–30 min.
• Run: 80–150 V, typically 30–90 min, 4–10°C for optimal resolution.

Note: Detection workflows (chemiluminescent vs fluorescent) depend on probe labeling, which is not specified for this item.

Safety and Handling

• GHS Classification / Signal Word / Pictograms / H-Statements: Not specified for this item; refer to the SDS for authoritative safety information.
• Intended use: For research use only.

General laboratory safety (applicable to nucleic acid probes; literature/practice-based):

• PPE: Wear lab coat, nitrile gloves, and safety glasses. Handle solutions in clean, nuclease-free conditions to preserve probe integrity.
• Hygiene: Avoid ingestion and inhalation of powders/aerosols. Do not pipette by mouth. Wash hands after handling.
• Incompatibilities: Avoid strong oxidizers and nuclease contamination (DNase). Use nuclease-free water and dedicated pipette tips/tubes.
• Aerosols and spills: For aqueous spills, absorb with inert material; clean with appropriate lab disinfectant. If lyophilized, avoid dust formation; gently reconstitute to minimize aerosol generation.
• Waste: Dispose of according to institutional guidelines for non-hazardous biological/chemical waste unless local rules specify otherwise.
• First aid (overview): If in eyes/skin, rinse with water for several minutes. If inhaled, move to fresh air. If ingested, rinse mouth and seek medical advice per institutional policy.

Notes:

• Some commercial DNA probes may contain trace preservatives (e.g., sodium azide) in buffer; for this item, such details are not specified—consult the SDS/CoA.
• Always defer to the product-specific SDS for comprehensive hazard, exposure controls, and spill/accident response.

Solvent Selection

This product is a nucleic acid probe rather than a solvent; typical solvent-selection concepts do not directly apply. For practical use, focus on reconstitution and working buffer compatibility.

General/literature guidance for EMSA probes:

• Reconstitution media: Nuclease-free water or low-ionic-strength buffer (e.g., 10 mM Tris–HCl, pH 7.5–8.0, 0.1–1 mM EDTA, “TE”). High-salt reconstitution can hinder accurate OD260 readings and downstream binding equilibria.
• Working solutions: Prepare in the binding buffer composition planned for EMSA to minimize dilution artifacts; include carrier DNA (e.g., poly(dI•dC)) only in the binding reaction, not stock.
• Compatibility: Avoid organic solvents; they destabilize duplex DNA and protein binding. Glycerol (2–10%) is commonly included in binding mixes to raise density and stabilize complexes (literature).
• Annealing: If supplied as single strands, anneal in aqueous buffer (e.g., 10 mM Tris, 50 mM NaCl, pH ~7.5–8.0) using controlled heat–cool ramp; do not use alcohols or strong denaturants.

Comparison (literature):

• Water vs TE: Water maximizes versatility but offers less pH buffering and nuclease chelation; TE provides modest buffering and nuclease protection via EDTA but may chelate metal cofactors if present in protein preps.
• TBE vs TAE (running buffers): EMSA typically favors TBE for sharper bands of protein–DNA complexes; TAE is less commonly used for EMSA due to lower buffering capacity at high voltage.

Storage and Reconstitution

• Storage Conditions: Store at -20°C (per Product Data). Avoid repeated freeze–thaw cycles.
• Shipped In: Ice chest + ice pads (per Product Data).
• Reconstitution Protocol: Not specified for this item; refer to CoA/Spec Sheet.

General handling notes (not item-specific):

• Upon receipt, verify whether the product is lyophilized or in solution. If lyophilized, allow the container to equilibrate to room temperature in a desiccated environment before opening to minimize moisture uptake.
• Use nuclease-free water or appropriate low-salt buffer for reconstitution, if required; filter tips and nuclease-free tubes are recommended.
• Prepare single-use aliquots and store at -20°C (or as directed by the CoA) to prevent degradation.

For definitive guidance (stock concentration, buffer composition, stabilizers, shelf-life), always consult the CoA/Spec Sheet, as these details are not provided in this listing.

Structure and Identity

Brief description: This product is an EMSA (Electrophoretic Mobility Shift Assay) probe intended for studying NF-κB DNA–protein interactions in research applications.

• Product Name: EMSA Probe NF-κB (SKU: E745607)
• Category: Chemical and Biochemical Reagents (Research use only)
• Molecular Formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.
• CAS / CID: Not specified for this item; refer to CoA/Spec Sheet.
• InChIKey / SMILES: Not applicable/not specified for this item; refer to CoA/Spec Sheet.

Structural features (general/literature):

• Probe class: Typically a synthetic double-stranded DNA oligonucleotide harboring an NF-κB consensus binding motif; may be unlabeled or modified with a reporter (e.g., biotin or fluorophore) for detection in EMSA. Specific sequence and modifications are not provided here.
• 2D structure (conceptual): Two antiparallel nucleotide strands annealed to form a short duplex; central region contains an NF-κB recognition element; may carry 5′/3′ labels depending on format. Exact base composition and label, if any, are not specified for this item.

Important note:

• Sequence, length, labeling chemistry, and extinction coefficient are critical identity attributes for EMSA probes but are not specified in this listing. Please consult the Certificate of Analysis (CoA) or Specification Sheet for item-specific identity details.

Synthetic Utility

This product is not a synthetic building block or reagent in the classical organic chemistry sense. It does not participate in named reactions or serve as a protecting-group handle.

• For chemical synthesis workflows, this probe has minimal direct utility. Instead, it is an analytical/biochemical tool to assess NF-κB–DNA binding.
• If modified, the probe could be used as a capture reagent in pull-down assays or affinity workflows (research only), but such modifications and formats are not specified for this item.

For synthetic chemistry needs, consider browsing Aladdin Scientific’s catalog of small-molecule reagents, coupling agents, catalysts, and protecting groups.

Target Specificity

Item-specific target details (sequence motif, subunit preference, species applicability, epitope mapping) are not specified for this product. Please refer to the CoA/Spec Sheet for definitive information.

General reminder:

• EMSA probes for NF-κB are typically designed to contain a κB recognition element to capture NF-κB–DNA binding in research assays, but the exact consensus variant, flanking bases, and any sequence optimizations are critical for specificity and are not provided here.