EMSA Probe C/EBP - 1.75μM

Cat. No.: E745601

グレード＆純度 1.75μM

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

概要

Aladdin's EMSA Probe - C/EBP is a C/EBP consensus oligonucleotide designed for EMSA . This double-stranded oligonucleotide contains the well-recognized binding sequence for the CCAAT/enhancer binding protein (C/EBP), and can be used as a probe for EMSA. The nucleotide sequence of C/EBP 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.

仕様

仕様と純度

1.75μM

安定性と保管

Store at -20℃ for up to 1 year.

保管条件

Store at -20°C

入荷

Ice chest + Ice pads

この製品は冷冷この冷このこの製品の運送が必要です。地上およびその他の経済サービスは利用できません。

Product Line

生物製品

Application Protocols

Tested/validated applications, recommended dilutions, and positive controls are not specified for this item in the provided Product Data. Please refer to the product CoA/Spec Sheet and SDS for any item-specific protocol notes. General EMSA workflow guidance is provided under Reaction & Applications and Reaction Conditions as literature context only.

Biological Roles

General background on the intended target (from product name) — C/EBP transcription factors; not item-specific claims:

• C/EBP family (CCAAT/enhancer-binding proteins) are basic leucine zipper (bZIP) transcription factors that bind to CCAAT/enhancer motifs in gene promoters/enhancers.
• Members (e.g., C/EBPα, β, δ, γ, ε, ζ) regulate differentiation, metabolism, inflammation, and stress responses. Dimerization (homo/heterodimers) modulates DNA-binding specificity and transcriptional outcomes (literature).
• DNA recognition: C/EBPs bind palindromic or half-site motifs often summarized as a consensus containing “CCAAT” or closely related sequences; exact motif preference varies by isoform and partner dimer.
• Experimental relevance: EMSA with a C/EBP consensus probe is widely used to monitor nuclear translocation/activation, compare binding across treatments, or validate promoter interactions prior to reporter assays or ChIP (literature context).

Buffer Applications

Item-specific buffer composition is not provided. The following are literature-standard EMSA buffer concepts for C/EBP-family DNA binding:

• Binding buffer (typical starting point):
  • 10 mM Tris–HCl, pH 7.5–8.0; 50–150 mM KCl or NaCl; 1 mM DTT; 1 mM EDTA (optional/low); 2–10% glycerol; 0.05–0.2% NP-40/Tween-20 (optional, to reduce nonspecific interactions). Include poly(dI·dC) or salmon sperm DNA as nonspecific competitor.
• Divalent cations: C/EBP binding is generally tolerant to low Mg2+; excessive Mg2+ can stabilize nonspecific interactions—titrate as needed.
• Gel and running buffer:
  • Native PAGE (4–6% acrylamide for typical 20–30 bp probes) in 0.5×–1× TBE or 1× TGE. Pre-run gels to equilibrate temperature/ionic strength.
• Sample loading:
  • Maintain final glycerol ~5–10% for stable band entry; avoid dyes that interact with proteins/DNA unless validated for EMSA.
• Supershift conditions:
  • Add validated anti-C/EBP antibody after initial binding equilibration; avoid detergents or salts that disrupt Ab–TF–DNA complexes.

All values are literature guidance and not specifications for this item.

Green Alternatives

For EMSA detection, greener choices focus on avoiding hazardous reagents and minimizing waste. Item-specific labeling is not provided; the following are literature-based considerations:

• Non-radioactive detection over radioisotopes:
  • Biotin–streptavidin chemiluminescence or DIG–antibody systems avoid 32P/33P handling and radioactive waste.
  • Tradeoff: Slightly lower sensitivity than 32P in some systems; typically compensated by longer exposures or optimized conditions.
• Fluorescent probes:
  • Directly labeled oligos (e.g., FAM, Cy dyes) enable gel imaging without chemiluminescent substrates, reducing consumables and waste.
  • Tradeoff: Requires compatible imagers; photobleaching and dye–protein interactions may need evaluation.
• Buffer and gel considerations:
  • Use Tris–glycine or Tris–borate systems prepared from concentrated stocks to reduce packaging waste.
  • Minimize ethidium bromide; use safer nucleic acid stains if post-staining free probes (verify compatibility with protein binding).
• Process efficiency:
  • Optimize binding to reduce repeat runs; aliquot probes to extend shelf life and minimize discard due to degradation.

Pharmaceutical Uses

Not applicable. This product is intended for research use only (as stated in Product Data) and is not described for any pharmaceutical formulation or manufacturing role.

General note (context): Synthetic oligonucleotides can serve as analytical tools in process development or QC assays, but no such application is specified for this item.

Physical Properties

Item-specific physico-chemical specifications were not provided.

• State/form: Not specified for this item; refer to CoA/Spec Sheet (oligonucleotide probes are typically supplied lyophilized or in aqueous buffer).
• Concentration (if solution), OD260, A260/A280, extinction coefficient: Not specified for this item; refer to CoA/Spec Sheet.
• pH (if buffered), salt content, stabilizers: Not specified for this item; refer to CoA/Spec Sheet.

General/literature guidance for oligonucleotide probes (for context only):

• Oligos are highly water-soluble; typical stock preparation is 10–100 µM in nuclease-free water or 1× TE (10 mM Tris, 1 mM EDTA, pH ~7.5–8.0).
• UV absorbance maximum ~260 nm; concentration can be estimated by A260 and sequence-specific extinction coefficients (ε depends on base composition).
• Melting temperature (Tm) depends on length, GC content, and ionic strength; duplex formation typically favored by monovalent cations (e.g., 50–150 mM K+ or Na+). These are literature values and not item specifications.

Quality and Grades

• Grade/purity: Not specified for this item; refer to CoA/Spec Sheet.
• Appearance/form: Not specified for this item; refer to CoA/Spec Sheet.
• Category path: 全部 / 可售 / 化学和生化试剂.

How to interpret probe quality (general, for context):

• Oligonucleotide purification levels (literature): Desalt, cartridge, HPLC, or PAGE purification influence purity and background in EMSA. Higher-purity (e.g., HPLC) reduces smear/non-specific bands and improves sensitivity.
• Label quality: For biotin or DIG labels (not specified here), verified label incorporation and lot-specific OD260 can facilitate consistent loading. For fluorescent probes, spectral purity and dye:oligo ratio matter for quantitation.
• Lot-specific documentation: For EMSA probes, vendors often provide sequence, length, expected Tm, and recommended annealing conditions. These are not specified for this item; consult the CoA/Spec Sheet if available.
• Application suitability: “EMSA-grade” typically implies nuclease-free handling and low salt/contaminant backgrounds suitable for native PAGE detection (general guidance, not a specification of this item).

Reaction and Applications

This product is intended for electrophoretic mobility shift assays (EMSA) targeting C/EBP DNA-binding activity (inferred from product name). Item-specific sequence, label, or assay parameters are not provided.

General EMSA applications and considerations (literature):

• Purpose: Detect sequence-specific DNA binding of transcription factors. A C/EBP consensus probe enables visualization of C/EBP–DNA complexes as retarded bands on native PAGE.
• Specificity controls: Include competition with unlabeled specific probe, non-specific competitor (e.g., poly(dI·dC)), and supershift with anti-C/EBP antibody to confirm factor identity.
• Sample sources: Nuclear extracts, whole-cell lysates, or purified C/EBP proteins. Maintain low salt and native conditions to preserve binding.
• Binding mix components: Tris or HEPES buffer, KCl/NaCl (typically 50–150 mM), Mg2+ (0–5 mM), DTT (0.5–2 mM), glycerol (2–10%), and nonspecific competitor DNA; probe at low pM–nM range to avoid probe excess binding artifacts (all literature guidance).
• Detection: Depends on probe label (biotin/DIG/fluorophore/radioisotope). Label type for this item is not specified.
• Data quality tips: Verify duplex formation by annealing (if supplied single-stranded), avoid nuclease contamination, and titrate protein to stay within linear binding range.

Reaction Conditions

Item-specific assay conditions are not provided. The following are literature-standard EMSA binding and electrophoresis conditions for C/EBP probes:

• Probe preparation:
  • If supplied single-stranded, anneal complementary strands by heating to 90–95°C for 2–5 min, then slow-cool to room temperature in annealing buffer (e.g., 10 mM Tris, 50 mM NaCl, 1 mM EDTA). Verify duplex by native PAGE.
• Binding reaction (typical ranges):
  • Total volume: 10–20 µL; protein: titrate from 1–10 µg nuclear extract or 10–200 nM purified protein; probe: 10–100 pM (for labeled) to remain binding-limited. Incubate 15–30 min at room temperature or 4–10°C for tighter complexes.
  • Add nonspecific competitor DNA (e.g., 0.05–0.5 µg/µL poly(dI·dC) stock to achieve 50–200 ng per reaction) to reduce background.
• Competition/supershift:
  • Pre-incubate protein with 50–200× molar excess unlabeled specific or mutated competitor for 10–15 min; for supershift, add antibody (validated clone) and incubate an additional 10–20 min.
• Electrophoresis:
  • 4–6% native polyacrylamide in 0.5×–1× TBE; run at 4–10°C where possible to maintain complex stability. Voltage 8–12 V/cm until separation achieved (typically 1–2 h).
• Detection:
  • Depends on label (biotin/DIG/fluor/radio); follow the label-specific protocol. Label type for this item is not specified.

All parameters are literature guidance and not specifications for this item.

Safety and Handling

• GHS classification, pictograms, H-statements, signal word: Not specified for this item; consult the SDS.
• General laboratory PPE: Lab coat, nitrile gloves, and safety glasses recommended. Handle with clean, RNase/DNase-free technique to prevent contamination and degradation.
• Storage incompatibilities: Avoid nucleases, microbial contamination, and repeated freeze–thaw cycles. Keep away from strong oxidants and extreme pH unless compatible with probe chemistry (label chemistry, if any, not specified).
• Engineering controls: Use clean benches or PCR hoods when preparing binding reactions to minimize nuclease contamination.
• First aid (general): If skin or eye contact occurs with buffers or solutions, rinse with water. If swallowed or inhaled (e.g., dried powder dust), seek medical advice. Follow local institutional safety procedures.
• Special risks: None specific provided. If probe is radioactively labeled (not specified), follow radiation safety procedures; if biotin/chemiluminescent or fluorescently labeled (not specified), handle according to the label’s safety profile.
• Defer to the SDS for authoritative hazard, spill, and disposal guidance.

Solvent Selection

Not typically applicable. EMSA probes are nucleic-acid reagents intended for aqueous buffers rather than organic solvents.

General notes (literature):

• Use nuclease-free water or 1× TE for stock solutions; avoid organic solvents that can denature proteins or disrupt DNA–protein binding.
• Glycerol (2–10%) may be present in binding mixes to aid complex stability and sample loading; its use depends on assay design and is not specified for this item.

Storage and Reconstitution

• Storage conditions (item-specific): Store at -20°C (per Product Data). Minimize freeze–thaw cycles. Shipped in ice chest with ice pads.
• Stability: Not specified for this item; refer to CoA/Spec Sheet. As general practice, oligonucleotide probes are stable for months at −20°C when protected from nucleases and light (if dye-labeled).
• Reconstitution (general best practice; not item-specific):
  • Spin down briefly before opening. Reconstitute lyophilized material in nuclease-free water or 1× TE to a convenient stock (e.g., 10–100 µM). If supplied in solution, verify concentration by A260 if sequence/ε are available.
  • For duplex probes supplied as single strands, anneal complementary strands by heat/slow-cool in low-salt buffer as outlined under Reaction Conditions.
• Aliquoting:
  • Prepare single-use aliquots to avoid repeated freeze–thaw. Store working aliquots at −20°C; short-term (≤24 h) storage at 4°C may be acceptable for daily use.
• Handling precautions:
  • Use DNase/RNase-free plastics and tips; wear gloves; avoid microbial contamination. Protect from light if fluorescently labeled (label not specified for this item).

Structure and Identity

• Item type: Nucleic-acid–based probe for electrophoretic mobility shift assay (EMSA), intended for DNA–protein binding studies involving C/EBP transcription factors (inferred from product name; sequence/label not provided).
• 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 applicable/Not specified for this item (oligonucleotide probe; exact composition not disclosed).
• Structural features (general, literature):
  • EMSA probes are commonly short double-stranded DNA oligonucleotides harboring a transcription-factor consensus sequence (here, C/EBP consensus such as 5′-TTNNGCAAT-3′ motif; literature example only).
  • May be supplied unlabeled or with a reporter tag (e.g., biotin, digoxigenin, or fluorophore) for detection after native PAGE; labeling for this item is not specified.
• 2D description (general): Duplex DNA with defined binding motif flanked by stabilizing bases; ends may be blunt or overhangs depending on design (not specified for this item).

Synthetic Utility

Not applicable. EMSA probes are analytical/biochemical reagents used to monitor DNA–protein interactions, not building blocks or reagents for chemical synthesis. For practical use, refer to Reaction & Applications and Buffer Applications sections.

Target Specificity

• Intended target (from product name): C/EBP transcription factor DNA-binding activity. No further target details (isoform, species reactivity, consensus sequence, or antibody clone for supershift) are provided in the Product Data.
• Sequence, motif, and any cross-reactivity information: Not specified for this item; refer to CoA/Spec Sheet.

Only the product name indicates the C/EBP context; no additional specificity claims are made here.