EMSA Probe CREB - 1.75μM

Cat. No.: E745603

GRADE & PURITY 1.75μM

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Overview

Aladdin's EMSA Probe - CREB is a CREB consensus oligonucleotide designed for EMSA . This double-stranded oligonucleotide contains the well-recognized binding sequence for cyclic-AMP response binding proteins (CREB), and can be used as a probe for EMSA. The nucleotide sequence of CREB 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 .For detailed operation of EMSA, please refer to the manual of our EMSA kit.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

Specifications & Purity

1.75μM

Stability And Storage

Store at -20℃ for up to 1 year.

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

This product requires cold chain shipping. Ground and other economy services are not available.

Application Protocols

Tested applications, recommended dilutions, and positive controls for this specific item: Not specified for this item; refer to CoA/Spec Sheet.

Note: While generic EMSA workflows are described under Reaction Conditions and Buffer Applications, definitive, item-specific protocol parameters (probe concentration, annealing instructions, labeling/detection steps, and control recommendations) must be taken from the manufacturer’s datasheet for SKU E745603.

Biological Roles

Context (literature/general): The EMSA Probe CREB is intended to interrogate the interaction between CREB (cAMP response element-binding protein) and its cognate DNA element (the CRE). CREB is a widely expressed bZIP transcription factor that binds the palindromic consensus sequence 5′-TGACGTCA-3′ to regulate transcription in response to intracellular signaling (e.g., cAMP/PKA, Ca2+/CaMK pathways).

• DNA–protein interaction: CREB dimerizes and binds the CRE motif; DNA binding can be modulated by CREB phosphorylation (e.g., at Ser133) and by cofactors such as CBP/p300.
• Gene regulatory context: CRE motifs are found in promoters/enhancers of genes involved in metabolism, plasticity, and stress responses; EMSA provides a biochemical readout of sequence-specific binding.
• Probe function: The probe itself has no intrinsic biological activity; it serves as a reporter duplex to capture CREB–DNA complex formation for separation on native polyacrylamide gels.

Item-specific biological attributes (sequence, modifications, length) that could influence affinity, specificity, and detection are not specified for this item and should be verified in the CoA/Spec Sheet before experimental design.

Buffer Applications

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

General EMSA buffer systems (literature guidance):

• Binding buffer (typical): 10 mM Tris-HCl pH 7.5–8.0, 50–100 mM KCl/NaCl, 1 mM DTT, 1 mM EDTA, 5–10% glycerol. Include 0.05–0.1 mg/mL poly dI·dC (or salmon sperm DNA) to suppress nonspecific binding.
• Metal/cofactor considerations: Some DNA-binding proteins require divalent cations (e.g., Mg2+). If so, reduce or omit EDTA accordingly; optimize ionic strength for specificity versus affinity.
• Gel running buffer: 0.5× TBE or 0.25× TBE commonly used for native PAGE to limit heating and preserve complexes.
• Loading dye: Glycerol-based without SDS; tracking dyes such as bromophenol blue at very low concentration to avoid interference.

Practical tips:

• Maintain reactions on ice until the binding incubation (often room temperature or 25–30 °C) to reduce nonspecific interactions.
• Titrate salt and glycerol to sharpen bands; excessive salt weakens specific binding.
• For fluorescently labeled probes, avoid quenchers and minimize photobleaching by protecting from light.

These recipes are provided as general guidance; validate and adjust for your specific protein preparation and detection modality.

Green Alternatives

Greener choices relate primarily to detection chemistry and waste profile rather than solvent selection for EMSA probes.

• Non-radioactive detection (greener) vs 32P radiolabeling:

  • Non-radioactive systems (e.g., biotin–streptavidin HRP, DIG–anti-DIG, or fluorescent labels) minimize radiological hazards and specialized waste, with high sensitivity and longer probe shelf life.
  • Trade-offs: Slightly lower sensitivity than high-specific-activity 32P in some setups; may require chemiluminescent substrates and imaging systems.

• Aqueous-only workflows:

  • Use aqueous buffers and avoid organic solvents or halogenated waste during gel preparation and running. Polyacrylamide gels and TBE/TAE buffers are standard and have manageable waste profiles when used without radionuclides.

• Consumables and process efficiency:

  • Prepare concentrated probe stocks and aliquot to minimize repeated syntheses/shipments.
  • Employ reusable gel rigs and minimize single-use plastics where sterility is not critical.

Comparison (general):

• 32P radiolabel EMSA: highest sensitivity; requires radiation controls, generates long-lived waste.
• Biotin/chemiluminescent EMSA: excellent sensitivity, safer handling, routine imaging; minimal hazardous waste.
• Fluorescent EMSA: multiplexing possible; requires compatible scanners, potential photobleaching.

For SKU E745603, the labeling chemistry, if any, is not specified; consult the CoA/Spec Sheet to select the most sustainable compatible detection method.

Pharmaceutical Uses

This product is a research-use nucleic acid probe for biochemical assays (EMSA). It is not an excipient, API, or diagnostic. No pharmacopeial status is implied.

• Typical role: laboratory tool for characterizing transcription factor–DNA interactions.
• Manufacturing/formulation relevance: none in standard pharmaceutical formulation workflows.

Item-specific regulatory or compendial information: Not specified for this item; refer to CoA/Spec Sheet.

Research Use Note: For research use only.

Physical Properties

Item-specific physical properties (specifications):

• Appearance, concentration, solvent, and extinction coefficient: Not specified for this item; refer to CoA/Spec Sheet.
• Melting point (Tm, duplex), pH of solution, and OD260/OD280 ratio: Not specified for this item; refer to CoA/Spec Sheet.

Literature/general properties for EMSA DNA probes (context only):

• State: typically provided lyophilized or in an aqueous buffer (e.g., TE or water). Duplex probes are often annealed prior to use.
• Solubility: water-soluble; compatible with low-ionic-strength buffers used in DNA–protein binding reactions.
• Duplex thermal behavior: probe Tm depends strongly on sequence, length, GC content, and salt. Short CRE-containing duplexes (16–30 bp) commonly have Tm values ranging from ~45–75 °C (literature) under standard saline conditions.
• UV absorbance: strong at 260 nm; quantitation via A260 using sequence-specific extinction coefficients (literature).
• Stability: dry, desiccated storage at −20 °C supports long shelf life; aqueous solutions are stable refrigerated/−20 °C with limited freeze–thaw (literature).

Note: Any numerical property (e.g., concentration, Tm, OD260) for SKU E745603 must be obtained from the product’s CoA/Spec Sheet; none are specified here.

Quality & Grades

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

Context on quality for EMSA oligonucleotide probes (literature/general):

• Common purification levels: Desalted, HPLC-purified, or PAGE-purified. EMSA benefits from higher-purity duplexes to minimize truncated species that can complicate banding patterns.
• Analytical controls typically include: OD260 quantitation, mass confirmation (e.g., MALDI-TOF/ESI), and if duplexed, verification of annealing (native PAGE or melting curves).
• Low background detection: For chemiluminescent/biotin systems, ensure minimal free label and confirm high specific activity for radiolabeled probes.
• Stabilizers: Some suppliers include Tris/EDTA (TE) for pH buffering and nuclease inhibition. If stabilizers or carrier nucleic acids are present for this item, they would be listed on the CoA.

How grade impacts use:

• HPLC/PAGE purification yields sharper EMSA bands and reduces nonspecific interactions.
• Stringent QC supports reproducibility in competition assays and supershift experiments.

For SKU E745603, consult the CoA/Spec Sheet for the exact purification grade, sequence verification method, and any stabilizers/excipients used.

Reaction & Applications

Manufacturer application (verbatim): Not provided.

General/literature applications for a CREB EMSA probe:

• Electrophoretic Mobility Shift Assay (EMSA/Gel Shift): Detect and characterize CREB–DNA complexes using a double-stranded CRE-containing probe. Mobility shifts indicate binding; specificity is tested by competition (specific vs nonspecific competitor) and supershift with an anti-CREB antibody.
• Transcription factor activity profiling: Compare nuclear extracts from stimulated vs control cells to assess CREB activation (e.g., via phosphorylation-dependent DNA binding; analysis remains biochemical, not clinical).
• Binding affinity assessments: Estimate apparent Kd by titrating protein over fixed probe; analyze band intensities (literature guidance).
• Protein–DNA interaction mapping: Evaluate effects of sequence mutations around the core TGACGTCA motif on CREB binding.

Practical tips (literature):

• Use freshly prepared nuclear extracts; keep reactions on ice prior to binding incubation.
• Include poly dI·dC (or salmon sperm DNA) to reduce nonspecific binding.
• Maintain low salt and include 1 mM DTT and 0.05–0.1% NP-40/Tween-20 only if compatible with the protein.
• Optimize probe concentration (typically low pM–low nM) to remain below protein concentration in affinity determinations.

Note: The exact label type, sequence, and recommended detection method for SKU E745603 are not specified here. Consult the CoA/Spec Sheet for item-specific application parameters.

Reaction Conditions

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

General EMSA conditions (literature guidance for CREB–CRE binding):

• Binding mix: duplex CRE probe at low pM–nM; protein (purified CREB or nuclear extract) at variable concentrations; 10 mM Tris-HCl pH 7.5–8.0, 50–100 mM KCl/NaCl, 1 mM DTT, 1 mM EDTA, 5–10% glycerol, ±0.05–0.1 mg/mL poly dI·dC.
• Incubation: 20–30 min at room temperature (20–25 °C); some protocols use 15 min on ice followed by 10 min at RT. Keep protected from light for fluorescent probes.
• Gel: 4–6% native polyacrylamide (29:1 acrylamide:bis), pre-run 15–30 min in 0.5× TBE. Run at 4–10 V/cm, typically 4 °C to stabilize complexes.
• Controls: specific competitor (100× unlabeled CRE duplex); nonspecific competitor; supershift with anti-CREB antibody to confirm identity of the shifted complex.
• Detection: depends on label (chemiluminescence for biotin/DIG; fluorescence imaging for dye labels; autoradiography or phosphorimaging for 32P).

Expected outcomes (literature): clear free-probe band and one or more retarded bands for protein–DNA complexes; supershifted band upon antibody addition. Optimize salt and probe levels to preserve resolution and specificity.

Safety & Handling

Regulatory/SDS information for this specific item:

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

General guidance for oligonucleotide EMSA probes (non-hazardous laboratory reagent context):

• Expected hazards: Unlabeled DNA oligonucleotides are generally considered low hazard, but handle using good laboratory practice. If labeled (e.g., biotin or fluorophore), hazards are typically minimal; if radioactively labeled, follow institutional radiation safety procedures.
• PPE: Lab coat, gloves, and safety glasses. Avoid aerosol generation; work on clean benches to prevent nuclease contamination.
• Incompatibilities: Avoid nucleases (DNase), strong oxidizers, and extreme pH. Use nuclease-free tips/tubes and dedicated buffers.
• First aid (general): In case of contact with eyes/skin, rinse with water. If inhaled (powder/aerosol), move to fresh air. If ingested, rinse mouth; seek medical advice as per institutional policy. Defer to SDS for authoritative instructions.
• Special risks and handling tips:
  • Use nuclease-free water/buffers and sterile, low-retention plasticware.
  • For radioactive probes, maintain shielding, monitoring, and waste segregation.
  • For fluorescent probes, minimize light exposure; wrap tubes in foil.

Waste disposal: Dispose according to local regulations. Radioactive or chemiluminescent detection wastes require designated streams per institutional EH&S.

Solvent Selection

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

General guidance for EMSA DNA probes:

• Primary medium: Aqueous buffers. Typical reconstitution in nuclease-free water or TE (10 mM Tris-HCl, 0.1–1 mM EDTA, pH 7.5–8.0).
• Binding reaction buffers: low to moderate ionic strength (e.g., 10 mM Tris, 50–100 mM KCl/NaCl, 1 mM DTT, 1 mM EDTA), sometimes 5–10% glycerol to aid complex stability and gel loading (literature).
• Avoid: High concentrations of chaotropes, detergents, or organic solvents that can disrupt protein–DNA interactions. Trace amounts of carrier (e.g., poly dI·dC) are often included to suppress nonspecific binding (literature).

Selection notes:

• Use water for initial dissolution; switch to working buffer for annealing and storage aliquots.
• For fluorescently labeled probes, maintain buffers free of primary amines if NHS-ester labeling is anticipated; protect from light.

Comparison (general):

• Water vs TE: Water maximizes versatility; TE provides better long-term stability (EDTA chelates adventitious metal ions) but can modestly affect some metalloproteins. Choose based on the DNA-binding protein’s cofactor requirements.

For SKU E745603, the recommended reconstitution/storage buffer, if any, will be listed on the CoA/Spec Sheet.

Storage & Reconstitution

Item-specific instructions:

• Storage conditions: Store at −20 °C (per Product Data). Minimize freeze–thaw cycles by aliquoting upon first use.
• Shipping: Shipped in ice chest + ice pads (per Product Data).
• Reconstitution buffer, concentration, and stabilizers: Not specified for this item; refer to CoA/Spec Sheet.

General guidance (literature):

• Upon receipt, briefly centrifuge to collect contents. If lyophilized, reconstitute with nuclease-free water or TE (10 mM Tris-HCl, 0.1–1 mM EDTA, pH 7.5–8.0) to a convenient stock (e.g., 10–100 µM). If supplied single-stranded, anneal complementary strands by heating to 90–95 °C for 2–5 min and slowly cooling to room temperature, then to 4 °C.
• Prepare single-use aliquots; store working stocks at −20 °C (weeks–months) and avoid repeated freeze–thaw. For fluorescently labeled probes, protect from light (foil-wrapped tubes) and store in amber vials where possible.
• Thaw on ice, mix gently, and keep on ice during setup to limit nuclease activity. Use low-retention, nuclease-free consumables.

Always consult the CoA/Spec Sheet for any item-specific instructions that supersede the general guidance above.

Structure & Identity

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

• Product type: Nucleic acid probe for EMSA (Electrophoretic Mobility Shift Assay) targeting the CREB DNA-binding site.
• CAS, CID, InChIKey, SMILES: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular formula and molecular weight: Not specified for this item; refer to CoA/Spec Sheet.

Literature/general description (for context):

• EMSA probes for CREB (cAMP response element-binding protein) are typically short, double-stranded DNA oligonucleotides containing a CRE consensus motif 5′-TGACGTCA-3′, optionally with flanking bases to enhance binding and gel resolution.
• Probes may be supplied unlabeled or with reporter tags (e.g., biotin, DIG, fluorescent dyes, or 32P) for detection. The presence/absence and type of label for this specific item are not specified.
• 2D structural features (general): a linear, double-helical nucleic acid duplex comprising phosphodiester backbone, deoxyribose sugars, and nucleobases (A, T, G, C). The CRE motif introduces a palindromic core that supports high-affinity CREB bZIP domain binding.

Important: The exact sequence, length, strand stoichiometry, and labeling chemistry for SKU E745603 are not provided here; consult the item’s CoA/Spec Sheet for definitive identity details.

Synthetic Utility

Not typically applicable. EMSA Probe CREB is a functional biochemical reagent rather than a building block for chemical synthesis.

General/literature perspective:

• In molecular biology workflows, such probes support the design and validation of sequence-specific binding assays, promoter analyses, and structure–function studies (e.g., mutated CRE variants to map binding determinants).
• They can be used to benchmark binding kinetics and to calibrate alternative platforms (e.g., fluorescence anisotropy, SPR) by providing a validated DNA target sequence.

For organic synthesis or classical reaction chemistry, there is no direct utility; see Reaction & Applications and Buffer Applications for relevant usage.

Target Specificity

Item-specific details (antigen/epitope/sequence/label): Not specified for this item; refer to CoA/Spec Sheet.

General context (for understanding only): CREB binds the CRE consensus 5′-TGACGTCA-3′ as a dimer via its bZIP domain. EMSA probes designed with this motif and appropriate flanking bases typically yield high-specificity complexes with CREB.

No claims regarding sequence, label, or tested specificity are made for SKU E745603 in the absence of product data.