EMSA Probe AP2 - 10μM

Cat. No.: E745598

GRADE & PURITY 10μM

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

개요

Aladdin's EMSA Probe – AP2 is an AP2 consensus oligonucleotide designed for EMSA . This double-stranded oligonucleotide contains the well-recognized binding sequence for AP2 transcription factors, and can be used as a probe for EMSA. The nucleotide sequence of AP2 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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Application Protocols

No item-specific, validated protocol is provided for EMSA Probe AP2. The following is a general, literature-based workflow for non-radioactive EMSA using an AP-2 probe.

• Probe preparation (general):

  1. Reconstitute complementary oligos in nuclease-free water or 1× TE. 2) Mix equimolar strands, heat to 90–95 °C for 2–5 min, and slowly cool to room temperature to anneal. 3) Quantify by A260.

• Binding reaction (10–20 µL total):

  • 10 mM Tris-HCl or HEPES (pH 7.5–8.0), 50–100 mM KCl/NaCl, 1 mM DTT, 5–10% glycerol, 50–200 ng/µL poly dI·dC, ±0.05% NP-40.
  • Add protein (nuclear extract 1–10 µg or recombinant AP-2 as available) and incubate 10 min.
  • Add labeled probe (10–100 fmol) and incubate 15–30 min at RT.

• Competition/supershift (optional):

  • Preincubate with 50–200× unlabeled competitor or specific antibody 10–20 min before adding probe.

• Electrophoresis:

  • Load on 4–8% native PAGE in 0.5×–1× TBE. Run 8–12 V/cm for 45–90 min. Keep gel apparatus cool if complexes are labile.

• Transfer and detection (non-radioactive example):

  • For biotinylated probes: transfer to nylon membrane, UV crosslink, block, incubate with streptavidin–HRP, and visualize by chemiluminescence. For fluorescent probes: directly image on a gel scanner.

• Controls:

  • No-protein control (free probe), nonspecific competitor, mutant competitor, and supershift with AP-2 antibody.

Adjust reagent amounts and times to your sample; consult CoA/Spec Sheet for any item-specific recommendations (not specified here).

Biological Roles

This section summarizes general, literature-based biology of AP-2 transcription factor binding sites; it does not describe item-specific properties.

• AP-2 family overview (general/literature):

  • AP-2 (Activating Protein-2) refers to a family of sequence-specific transcription factors (e.g., TFAP2A/AP-2α, TFAP2B, TFAP2C, TFAP2D, TFAP2E) characterized by a basic helix–span–helix DNA-binding domain and dimerization capability.
  • They regulate gene expression programs in development, differentiation, and cellular stress responses by binding conserved GC-rich motifs.

• DNA recognition (general/literature):

  • AP-2 commonly recognizes palindromic/near-palindromic GC-core elements. Probes centered on these motifs enable in vitro detection of AP-2–DNA complexes.

• Functional context (general/literature):

  • AP-2 factors interact with co-regulators to modulate chromatin accessibility and transcriptional initiation.
  • DNA binding is influenced by local chromatin context, CpG methylation state, flanking sequence, and post-translational modifications on AP-2 proteins.

• Relevance of EMSA probes (general/literature):

  • EMSA probes model the minimal binding site, allowing isolation of sequence-specific binding from chromatin effects.
  • Competition with mutant or methylated variants can dissect contributions of core and flanking bases, or epigenetic modifications, to AP-2 affinity.

Note: This information is provided for background. The specific sequence, affinity, and validation data for EMSA Probe AP2 are not specified for this item; refer to CoA/Spec Sheet where available.

Buffer Applications

Nucleic acid EMSA relies on carefully formulated aqueous buffers. Item-specific buffer recommendations are not provided; the following are general, literature-based examples.

• Typical binding buffer (general example):

  • 10 mM Tris-HCl, pH 7.5–8.0 (or 10 mM HEPES, pH 7.5)
  • 50–150 mM KCl or NaCl
  • 1 mM DTT (reducing environment for protein stability)
  • 0.05–0.1% NP-40 or Tween-20 (optional)
  • 5–10% glycerol (enhances complex stability and sample loading)
  • 50–200 ng/µL poly dI·dC (nonspecific competitor)

• Electrophoresis buffer (general example):

  • 0.5×–1× TBE or 0.25×–0.5× TBE for native PAGE (lower ionic strength can sharpen complexes and minimize heating). Gel concentration: 4–6% for larger complexes; 6–8% for smaller complexes.

• Sample preparation (general):

  • Keep on ice; assemble protein last. Incubate 15–30 min at RT or 4–25 °C. Add loading dye lacking SDS and with minimal EDTA to avoid chelating essential metal ions required by some DNA-binding proteins.

• Supershift conditions (general):

  • Pre-incubate extract with specific antibody 10–20 min before adding probe; adjust glycerol and salt to maintain complex integrity.

• Notes:

  • Mg2+ is generally avoided unless required by the protein; EDTA in TE can help chelate trace metals that catalyze DNA degradation.
  • Optimize salt in small increments to balance specificity and complex stability.

Item-specific buffer guidance is not specified; refer to CoA/Spec Sheet if provided.

Green Alternatives

As an aqueous nucleic acid probe, environmental impact is primarily associated with buffer components and detection chemistry rather than organic solvent use.

• Greener choices within EMSA workflows (general/literature):
  • Prefer non-radioactive labels (biotin or fluorophores) over isotopes to reduce radiological hazards and waste streams while maintaining sensitivity.
  • Use Tris/HEPES buffers at moderate ionic strength; avoid halogenated solvents entirely (not required).
  • Replace ethidium bromide with safer nucleic acid stains when applicable.
  • Minimize single-use plastics by planning aliquots and using reusable gel rigs with long-life electrodes.

Comparison (general):

• Radioisotopic probes vs non-radioactive probes:
  • Sensitivity: 32P traditionally highest; modern biotin/fluorescent systems approach comparable sensitivity with optimized protocols.
  • Waste/safety: Radioisotopes demand specialized handling and disposal; non-radioactive systems reduce environmental and compliance burden.
  • Workflow: Non-radioactive methods are faster and do not require darkroom/film; they rely on chemiluminescence or fluorescence imagers.

Trade-offs:

• Non-radioactive probes may require longer exposure or optimized blocking/detection reagents.
• Certain fluorophores can photobleach; protect from light and store appropriately (labeling for this item is not specified).

Overall: For EMSA Probe AP2, choose non-radioactive detection where feasible and optimize buffer conditions to minimize reagent use and repeat runs.

Pharmaceutical Uses

This product is supplied for research use only.

• Role in pharmaceutical R&D (general/literature):

  • EMSA probes are used in discovery-stage research to study DNA–protein interactions, inform target validation, and profile transcription factor activities in cell or tissue extracts.
  • They can support assay development or screening of modulators that influence DNA-binding activity in vitro, as part of broader workflows.

• Excipient/formulation status:

  • Not applicable. Oligonucleotide probes are not pharmaceutical excipients and are not intended for formulation into drug products.

• Pharmacopeial status:

  • No pharmacopeial monograph is applicable to this research reagent (general). Any quality benchmarks are laboratory/QC specific.

• Compliance and use restrictions:

  • Research Use Only (RUO); not for human or animal therapeutic, diagnostic, or clinical use.

No item-specific pharmaceutical or manufacturing uses are specified for EMSA Probe AP2.

Physical Properties

Item-specific physicochemical specifications (exact MW, molar absorptivity, length, labeling dye, extinction coefficient) are not provided for this product.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.
• Solubility (general/literature):
  • Oligonucleotides are freely soluble in nuclease-free water and common low-ionic-strength buffers (e.g., 1× TE, 10 mM Tris, 1 mM EDTA, pH 7.5–8.0).
  • Solubility can be improved by gentle heating (≤55 °C) and brief vortexing/spin-down.
• pKa/logP/RI: Not applicable for nucleic acid polymers in the conventional sense; values are seldom reported as single numbers.
• Melting temperature, Tm (general/literature):
  • Depends on length, GC content, ionic strength, and sequence. Duplex EMSA probes of 18–30 nt typically exhibit Tm values well above ambient under standard binding buffer salt (literature). Exact Tm for this probe is not specified.
• UV absorbance (general/literature):
  • DNA oligos characteristically absorb at 260 nm; A260 can be used to estimate concentration using an extinction coefficient derived from sequence. Extinction coefficient for this item is not specified; refer to CoA if provided.
• Storage stability (see Storage tab for item-specific conditions):
  • Stability improves at −20 °C in low-salt, slightly basic buffers; repeated freeze–thaw should be minimized (general/literature).

Quality and Grades

Item-specific grade/purity and purification method are not provided for this product.

• Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.
• Typical quality options for EMSA probes (general/literature):
  • Desalted, PAGE-purified, or HPLC-purified oligos. EMSA generally benefits from PAGE or HPLC purification to ensure sharp bands and reduce truncated species.
  • Labeling quality (biotin, fluorophore, or radiolabel) is critical for detection sensitivity; specific label for this item is not specified.
• QC metrics commonly reported (general):
  • OD260 and calculated nmol/µg.
  • Analytical HPLC/PAGE purity.
  • MALDI-TOF/ESI-MS for mass confirmation.
  • Duplex annealing verification by native PAGE or UV-melting profile.
• Implications for use (general):
  • Higher-purity probes reduce nonspecific bands and smearing in EMSA.
  • Consistent lot-to-lot performance depends on controlled synthesis, deprotection, and purification processes; consult CoA for this item’s lot-specific metrics.
• Stabilizers/additives: Not specified for this item; refer to CoA/Spec Sheet.
• Documentation:
  • For regulated workflows or publication, retain CoA/SDS, including sequence, length, modifications, and purification data where applicable.

Reaction and Applications

Use context: EMSA Probe AP2 is intended for electrophoretic mobility shift assay (EMSA) studies. Item-specific application validation is not provided; the following is general literature guidance.

• EMSA principle (general/literature):
  • DNA–protein complexes migrate more slowly than free DNA in native polyacrylamide gels. A labeled AP-2 motif–containing probe highlights sequence-specific binding by AP-2 family transcription factors.
• Typical applications (general):
  • Assessing AP-2 binding activity in nuclear extracts or with recombinant protein.
  • Competition EMSA with excess unlabeled wild-type vs mutant competitor to demonstrate specificity.
  • Supershift assays with AP-2 antibodies to confirm complex identity (antibodies not included).
  • Mapping binding sites and effects of nucleotide substitutions flanking the AP-2 core element.
• Practical tips (general):
  • Anneal complementary strands prior to use; confirm duplex formation by native PAGE if necessary.
  • Optimize salt (50–150 mM KCl/NaCl) and glycerol (5–10%) to balance specificity and complex stability.
  • Include nonspecific competitor DNA (e.g., poly dI·dC) to suppress nonspecific binding.
  • Keep reactions on ice during setup; incubate binding at room temperature or 4–25 °C for 15–30 min (optimize per protein).
• Detection (general):
  • Depending on labeling (not specified for this item), detection may be chemiluminescent (biotin–streptavidin-HRP), fluorescent, or autoradiographic.
• Limitations (general):
  • EMSA reports binding but not functional transcriptional outcomes; follow-up assays (reporter gene, ChIP) are often complementary.

Reaction Conditions

“Reaction” here refers to DNA–protein complex formation and native gel separation in EMSA. Item-specific validated conditions are not provided; the following are general, literature-based guidelines to begin optimization.

• Binding reaction (general starting point):

  • Final volume: 10–20 µL.
  • Buffer: 10 mM Tris-HCl or HEPES (pH 7.5–8.0), 50–100 mM KCl/NaCl, 1 mM DTT, 5–10% glycerol, ±0.05% NP-40, 50–200 ng/µL poly dI·dC.
  • Probe: 10–100 fmol labeled duplex DNA per reaction (exact amount depends on label and detection sensitivity).
  • Protein: Titrate nuclear extract (1–10 µg) or recombinant AP-2 (0.1–1 µg) to achieve a clear shifted complex without overloading (literature ranges).
  • Incubation: 15–30 min at room temperature or 4–25 °C.

• Competition/supershift (general):

  • Competitor: 50–200× molar excess of unlabeled wild-type vs mutant probe, preincubated 5–10 min before adding labeled probe.
  • Supershift: Add specific antibody and incubate 10–20 min prior to probe addition.

• Electrophoresis (general):

  • Gel: 4–8% native polyacrylamide in 0.5×–1× TBE; pre-run 10–15 min to equilibrate.
  • Temperature: Run at 4–10 °C for labile complexes or room temperature for robust complexes.
  • Voltage/time: 8–12 V/cm for 45–90 min, adjusting for gel size and resolution.

• Detection (general):

  • Depends on label (not specified for this item): chemiluminescence (biotin-HRP), fluorescence imaging, or autoradiography.

These parameters serve as a starting framework; optimize per extract, protein purity, and probe design.

Safety and Handling

Safety information specific to this product is not provided. Always consult the SDS for authoritative guidance.

• GHS/Signal Word/Pictograms: Not specified for this item; refer to SDS.
• General hazard profile (general/literature):
  • Synthetic DNA probes are typically of low acute toxicity and are not expected to be volatile; however, handle as a laboratory chemical. Some labeled probes may contain dyes or linkers with additional hazards (not specified here).
• PPE recommendations (good lab practice):
  • Lab coat, safety glasses, and nitrile gloves. Use nuclease-free technique to avoid contamination (RNase-/DNase-free consumables and tools).
• Handling notes (general):
  • Work on ice when preparing binding reactions for EMSA to limit nuclease activity.
  • Avoid microbial contamination; prepare and store aliquots in sterile, nuclease-free tubes.
• Incompatibilities (general):
  • Strong nucleases, high temperatures, and extreme pH can degrade oligonucleotides. Oxidizing agents may affect certain labels if present (labeling not specified for this item).
• First-aid overview (general):
  • Skin/eye contact: Rinse with water. Ingestion/inhalation: Seek medical attention as per institutional policy. No special antidotes; treat symptomatically. Defer to SDS.
• Waste: Dispose according to local regulations for non-hazardous aqueous laboratory waste unless SDS indicates otherwise.

Solvent Selection

This product is a nucleic acid probe and is used in aqueous buffers rather than organic solvents.

• Primary solvent system (general/literature):
  • Nuclease-free water or low-ionic-strength buffers (e.g., 1× TE: 10 mM Tris-HCl, 1 mM EDTA, pH 7.5–8.0).
  • EMSA binding buffers typically include Tris/HEPES, monovalent salts (KCl/NaCl), DTT, glycerol, and a nonspecific competitor (poly dI·dC).
• Miscibility/compatibility (general):
  • Compatible with standard aqueous buffers; avoid high concentrations of organic solvents which can destabilize DNA–protein interactions and alter electrophoretic mobility.
• When to choose water vs buffer (general):
  • Use nuclease-free water for initial resuspension; switch to storage in 1× TE to improve stability over time (EDTA chelates trace metal ions that catalyze degradation).
• Additives (general):
  • Low levels of glycerol (5–10%) can facilitate sample loading and protein stability in EMSA reactions.
• Non-applicability:
  • Organic solvent selection frameworks (e.g., polarity indices, dielectric constants) are not typically relevant to nucleic acid EMSA probes; focus on buffer composition and ionic strength instead.

Storage and Reconstitution

Item-specific storage conditions provided by the supplier:

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

Additional best practices (general/literature):

• Reconstitution:
  • Use nuclease-free water or 1× TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5–8.0). Target a convenient stock concentration (e.g., 10–100 µM) based on intended use; exact concentration is user-defined as item-specific guidance is not provided.
  • If duplex probe is supplied as single strands, anneal by heating to 90–95 °C for 2–5 min and slow cooling to room temperature.
• Aliquoting:
  • Prepare single-use aliquots to avoid repeated freeze–thaw cycles. Store working aliquots at −20 °C; for extended storage, −80 °C may further preserve integrity (general practice).
• Handling:
  • Thaw on ice, mix gently, and briefly spin down before use. Avoid vigorous vortexing of long oligos; brief, gentle mixing is sufficient.
  • Protect from light if the probe carries a photolabile or fluorescent label (labeling for this item is not specified).
• Stability:
  • Oligos are typically stable for months at −20 °C in TE buffer; avoid multiple freeze–thaw cycles and nuclease contamination. Item-specific shelf life is not specified; refer to CoA/Spec Sheet.

Note: Research use only. For definitive storage limits, concentration, and handling details of this SKU, consult the product CoA/SDS.

Structure and Identity

Brief overview: EMSA Probe AP2 is a nucleic-acid–based probe intended for electrophoretic mobility shift assay (EMSA) studies of AP-2 transcription factor binding. Item-specific molecular identifiers are not provided.

• Item-specific identifiers:

  • CAS: Not specified for this item; refer to CoA/Spec Sheet.
  • CID: Not specified for this item; refer to CoA/Spec Sheet.
  • InChIKey: Not applicable/Not specified for this item; refer to CoA/Spec Sheet.
  • SMILES: Not applicable for polymers/oligonucleotides; not specified for this item.
  • Molecular Formula: Not specified for this item; refer to CoA/Spec Sheet.
  • Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.

• Structural class (general/literature):

  • Likely a double-stranded DNA oligonucleotide containing an AP-2 transcription factor binding motif; some EMSA probes may be single-stranded to be annealed prior to use. Specific length, sequence, and modifications (e.g., 5′-biotin, fluorescent dye, radiolabel) are not specified for this item.

• Functional features (general/literature):

  • Contains a central AP-2 consensus element that facilitates sequence-specific binding by AP-2 family proteins.
  • May be used with unlabeled competitor or mutant probes in EMSA to verify specificity.

• 2D description (general/literature):

  • Short duplex DNA with canonical B-form geometry when annealed; central palindromic/near-palindromic AP-2 site flanked by stabilizing sequences to enhance binding and electrophoretic resolution. Sequence details are not provided for this specific product.

Synthetic Utility

This product is a functional biomolecular tool rather than a synthetic intermediate. Classical organic “synthetic utility” does not apply. The following describes its utility in experimental design (general/literature).

• Molecular tool utility:

  • Defined DNA sequences serve as reagents to interrogate sequence-specific binding of AP-2 transcription factors.
  • Mutagenesis series (systematic base substitutions) can be synthesized to map contributions of core and flanking bases to binding energy.

• Experimental design applications:

  • Allele-specific binding analysis by comparing wild-type vs variant AP-2 sites.
  • Methylation-sensitive EMSA by incorporating 5mC at CpG positions to test epigenetic effects on binding.
  • Competition binding assays to estimate apparent dissociation constants (Kd,app) under native conditions.

• Integration with other methods:

  • Use EMSA outcomes to guide reporter assays, ChIP, or CUT&RUN experiment design.
  • Probe-based pull-downs (with appropriate tags/labels) can enrich AP-2–associated complexes for downstream MS, provided capture chemistry is compatible (label for this item not specified).

Note: No item-specific synthetic chemistry applications are claimed for EMSA Probe AP2.

Target Specificity

Item-specific target validation data are not provided for EMSA Probe AP2. The following describes general, literature-based specificity principles for AP-2 EMSA probes.

• Intended target (general):

  • AP-2 family transcription factors (e.g., TFAP2A/B/C/D/E) that bind a conserved GC-rich DNA motif.

• Specificity controls (general):

  • Wild-type vs mutant competitor: Specific complexes are competed by excess unlabeled wild-type probe but not by mutant sequences lacking the AP-2 core.
  • Supershift: Addition of AP-2–specific antibody retards migration further, confirming complex identity.
  • Unrelated competitor DNA (e.g., SP1 site) should not displace AP-2–specific complexes at equal molar excess.

• Factors influencing specificity (general):

  • Flanking nucleotides and CpG methylation can modulate AP-2 affinity and apparent specificity.
  • Salt concentration and nonspecific competitor DNA levels strongly affect background and off-target binding.

• Cross-reactivity considerations (general):

  • Other GC-box–binding proteins may form complexes with certain sequences; robust competition and supershift controls are essential.

For probe sequence, length, label, and any lot-specific validation data, consult the CoA/Spec Sheet (not specified for this item).