EMSA/Gel-Shift Binding Buffer - 5X

Cat. No.: E745592

QUALITÄT & REINHEIT 5X

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Übersicht

EMSA/Gel-Shift Binding Buffer (5X) is the binding buffer for EMSA/Gel-Shift assay which is generally used to study protein-DNA interactions and to detect the activation level of transcription factors in cells. EMSA/Gel-Shift Binding Buffer (5X) can be used in EMSA for the binding of nuclear proteins or purified transcription factors to specific double-stranded oligonucleotides.This product contains active ingredients such as poly(dI-dC), DTT, glycerol, EDTA, NaCl, MgCl2 and Tris. The concentration of poly(dI-dC) has been optimized to eliminate non-specific bindings without affecting the specific binding between target transcription factor and the labeled probe.This product is not only suitable for isotope-labeled probes, but also for digoxin or biotin-labeled probes.This product is sufficient for 100 binding reactions between protein and probe.

Precautions：

All other reagents related to EMSA are not provided in this product.Please read the entire manual carefully before starting the experiment.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 following reactions by adding the reagents in sequence. Please refer to Figure 1 for the assay results. Negative Control ReactionNuclease-Free Water7µlEMSA/Gel-Shift Binding Buffer (5X)2µlNuclear Protein or Purified Transcription Factor0µlLabeled Probe1µlTotal Volume10µlCold Probe Competition ReactionNuclease-Free Water4µlEMSA/Gel-Shift Binding Buffer (5X)2µlNuclear Protein or Purified Transcription Factor2µlUnlabeled Probe 1µlLabeled Probe1µlTotal Volume10µl Super-shift ReactionNuclease-Free Water4µlEMSA/Gel-Shift Binding Buffer (5X)2µlNuclear Protein or Purified Transcription Factor2µlTarget Protein Specific Antibody1µlLabeled Probe1µlTotal Volume10µl Sample ReactionNuclease-Free Water5µlEMSA/Gel-Shift Binding Buffer (5X)2µlNuclear Protein or Purified Transcription Factor2µlLabeled Probe1µlTotal Volume10µl Cold Mutation Probe Competition ReactionNuclease-Free Water4µlEMSA/Gel-Shift Binding Buffer (5X)2µlNuclear Protein or Purified Transcription Factor2µlUnlabeled Probe1µlLabeled Probe1µlTotal Volume10µl2. Before adding the labeled probe, the reaction mix should be mixed well and incubated at room temperature (20-25℃) for 10 minutes to eliminate possible non-specific bindings between probes and proteins. Otherwise allow cold probes react preferentially. After adding the labeled probe, mix well and incubate at room temperature (20-25℃) for 20 minutes.3. Add 1μl of EMSA/Gel-Shift Loading Buffer (colorless, 10X) into each reaction, mix well and load the mixture immediately on a gel. Note: Sometimes bromophenol blue can affect protein-DNA interactions. It is recommended to use the colorless EMSA/Gel-Shift Loading Buffer, or that including a very small amount of blue loading buffer to make observation easier.4. Electrophoresis analysis:Figure 1. The typical EMSA result analyzed with ’s EMSA/Gel-Shift Kit . 1. Negative Control Reaction (labeled probe); 2. Sample Reaction (nucleoprotein containing activated target transcription factor + labeled probe); 3. Cold Probe Competition Reaction (nucleoprotein containing activated target transcription factor + labeled probe + unlabeled probe 100 times the amount of labeled probe); 4. Cold Mutant Probe Competition Reaction (nucleoprotein containing activated target transcription factor + labeled probe + unlabeled mutant probe 100 times the amount of labeled probe); 5. Super-shift Reaction (nucleoprotein containing activated target transcription factor + labeled probe + specific antibody of target transcription factor).

Spezifikationen

Spezifikationen & Reinheit

5X

Stabilität und Lagerung

Store at -20℃ for up to 1 year.

Storage

Store at -20°C

Verschickt in

Ice chest + Ice pads

Dieses Produkt erfordert Kühlkettenversand. Grundversand und andere Economy-Optionen sind nicht verfügbar.

Product Line

Biologische Produkte

Application Protocols

Tested applications, recommended dilutions, and positive controls are not specified for this item; refer to CoA/Spec Sheet and any included insert.

Note: General EMSA setup guidance is provided in the “Buffer Applications” tab for context only and should be optimized empirically for your system.

Biological Roles

This product facilitates the in vitro study of protein–nucleic acid interactions; it does not have a biological role in vivo. Context for users (general, literature):

• Transcription regulation: EMSA buffers enable detection of sequence‑specific binding of transcription factors to promoter/enhancer motifs, informing on DNA recognition elements.
• RNA interactions: Many RNA‑binding proteins (RBPs) recognize structured or motif‑containing RNAs; EMSA conditions can showcase affinity and complex stoichiometry.
• Cofactor dependence: Binding of some proteins depends on ions (e.g., Mg2+, Zn2+) or small molecules; buffers provide a controlled milieu to probe such dependencies.
• Nonspecific vs. specific binding: Ionic strength and competitor nucleic acids (e.g., poly(dI·dC)) modulate background binding, helping reveal true specificity.
• Thermodynamics/kinetics: By adjusting temperature, salt, and pH, users can assess Kd, cooperative binding, and competition—key biophysical descriptors for regulatory complexes.

Note: These are general biological study contexts. The buffer’s exact composition is not specified for this item and should be confirmed on the CoA/Spec Sheet for compatibility with the target protein and detection method. The product is intended strictly for laboratory research use.

Buffer Applications

Primary use: EMSA/Gel‑Shift Binding Buffer for assembling protein–DNA/RNA complexes prior to native PAGE.

General protocol guidance (literature; optimize per system; not product specifications):

• Working mix (example framework):
  • Probe (0.05–1 nM labeled oligo/RNA) + protein (titrate across 0.1–10× expected Kd).
  • Binding buffer at 1× final concentration.
  • Additives as required: 5–10% glycerol, 0.05–1 mM DTT/TCEP, 0–5 mM MgCl2/Zn2+, 0.005–0.05% nonionic detergent, poly(dI·dC) 10–100 ng/µL to suppress nonspecific binding.
  • Incubate 10–30 min at room temperature or on ice.
• Gel and running conditions:
  • Native PAGE (e.g., 4–6% for large complexes; 6–10% for small complexes) in 0.5×–1× TBE or appropriate running buffer.
  • Pre‑run gel 15–30 min to stabilize temperature and ionic environment.
  • Maintain gels cold (4–10 °C) for labile complexes or weak interactions.
• Loading and detection:
  • Include glycerol in samples for density; avoid dyes that interact with DNA/RNA unless validated.
  • Detect by autoradiography, fluorescence imaging, or streptavidin‑HRP/chemiluminescence for biotinylated probes.
• Controls:
  • Specific competitor (100× unlabeled probe), nonspecific competitor, supershift antibody, and mutant probe to confirm specificity.
• Troubleshooting:
  • Smearing: reduce glycerol/detergent, increase gel % or lower temperature.
  • Weak signal: decrease salt, add divalent ions if required, lengthen incubation, ensure probe integrity.

Item‑specific concentration, pH, and additives are not specified; consult the CoA/Spec Sheet for the exact usage instructions supplied with this SKU.

Green Alternatives

While a binding buffer has a modest environmental footprint, greener practice can still be applied.

• Strategy highlights (general):
  • Use ready‑to‑use reagents to cut repeat prep waste and minimize disposal of off‑spec batches.
  • Prefer non‑radioactive detection (fluorescent/biotin) when sensitivity permits to reduce radiological waste.
  • Consolidate runs and right‑size gel volumes to cut buffer consumption.

Comparison (general guidance; not product specifications):

| Option | Pros | Tradeoffs | |---|---|---| | Ready‑to‑use EMSA buffer (this product class) | Lot‑controlled, minimizes remake waste, consistent performance | Less flexibility to vary composition; cold‑chain shipment impact | | DIY EMSA buffer (from bulk salts) | Maximal compositional control; reduced shipping footprint | Higher operator time; potential batch variability and waste if out of spec | | Non‑radioactive probes | Safer handling; reduced hazardous waste | May require more material or enhanced imaging |

Additional tips:

• Use reusable gel rigs and minimize single‑use plastics by aliquoting wisely.
• If glycerol content is adjustable, use the minimum that maintains performance to reduce viscosity‑related rinsing and water usage.

Item‑specific eco attributes are not specified; consult the SDS and CoA for any information on preservatives or biocides.

Pharmaceutical Uses

This product is a research‑grade EMSA binding buffer intended for analytical studies of protein–nucleic acid interactions. It is not an active pharmaceutical ingredient or an excipient for clinical formulations.

• GMP/Pharmacopeia: Not specified for this item; refer to CoA/Spec Sheet if any compliance attributes are required.
• Process development (general, non‑clinical): EMSA can support preclinical research by characterizing DNA/RNA‑binding proteins and oligonucleotide interactions; however, the buffer is not intended for manufacturing or therapeutic use.

No clinical or therapeutic claims are made. For any regulated application, procure materials with appropriate certifications and change control.

Physical Properties

Item‑specific physical properties (pH, concentration, osmolality, viscosity, density, refractive index): Not specified for this item; refer to CoA/Spec Sheet.

General/literature expectations for EMSA binding buffers (for context only; not product specifications):

• Physical state: Aqueous solution (or concentrate) that is colorless to slightly yellow depending on components.
• Typical pH window (literature): ~7.0–8.5 depending on protein target and buffer system (e.g., Tris, HEPES, or phosphate). Optimal pH is protein‑dependent.
• Typical ionic strength (literature): 25–150 mM monovalent salt; often titrated to balance specificity vs. affinity.
• Typical additives (literature): 5–10% glycerol to aid complex stability and sample loading; 0.05–1 mM DTT/TCEP; 0–5 mM MgCl2 for factors that require divalent cations; 0.01% nonionic detergent.
• Solubility: Fully miscible with water; compatible with standard native PAGE running buffers (e.g., 0.5×–1× TBE/TGE) when appropriately diluted.

Boiling/melting points, vapor pressure, logP: Not applicable to a multicomponent aqueous buffer and not specified for this item.

Note: Always validate working pH, salt, and cofactor requirements empirically for the specific protein–nucleic acid system under study.

Quality and Grades

• Grade/purity: Not specified for this item; refer to CoA/Spec Sheet.
• What to expect from a dedicated EMSA/Gel‑Shift Binding Buffer (general guidance):
  • Consistent, lot‑to‑lot formulation to support reproducible protein–nucleic acid binding.
  • Low bioburden/low nuclease background; often prepared with nuclease‑free water and filtered. Verify on CoA if critical.
  • If provided as a concentrate (e.g., 5×/10×), clarity upon dilution and no salt precipitation at 4 °C are quality indicators.
• Stabilizers (if present): Reducing agents (e.g., DTT/TCEP) maintain protein activity but can degrade over time; check lot age and storage history. Trace detergents may reduce nonspecific binding and improve consistency.
• Documentation: For assay‑critical parameters (pH tolerance, salt range, compatible cofactors), consult the product’s CoA/Specification Sheet and SDS.
• Suitability: This buffer is intended for EMSA and related gel‑shift assays; for supershift assays or competition experiments, verify compatibility with antibodies and competitor nucleic acids.
• Compliance note: Life‑science reagent intended for laboratory research. Not manufactured to GMP unless explicitly stated on accompanying documents.

Reaction and Applications

Primary application: Electrophoretic Mobility Shift Assay (EMSA; gel‑shift) to assess and quantify protein–nucleic acid interactions under native conditions.

Key uses and considerations (general literature guidance):

• DNA‑ and RNA‑binding proteins: Transcription factors, RBPs, recombinases, and nucleases (catalytically inactive conditions). The buffer promotes specific complex formation prior to loading onto a native polyacrylamide gel.
• Specificity controls: Incorporate specific and nonspecific competitors (e.g., unlabeled consensus oligo; poly(dI·dC)) to distinguish specific binding from background.
• Supershift assays: Addition of antibodies targeting the protein can produce a supershifted band; ensure buffer compatibility with antibody binding (salt, detergent, and pH).
• Metal dependence: Some factors require Mg2+ or Zn2+; chelators like EDTA can disrupt complexes—titrate divalent ions carefully.
• Kinetics/thermodynamics: Salt titrations and temperature control (on ice or at room temperature) can modulate on/off rates and sharpen band resolution.
• Labeling modalities: Compatible with radiolabeled (32P), fluorescent, or biotinylated probes; choose detection and gel system accordingly.
• Practical tips:
  • Pre‑equilibrate components at the working temperature.
  • Incubate 10–30 min for many systems; adjust based on binding kinetics.
  • Use 5–10% glycerol for density/stability; avoid excessive glycerol that smears bands.
  • Keep ionic strength minimal for initial screens, then raise to challenge specificity.

Note: The exact formulation, concentrations, and pH of this item are not specified; optimize empirically for each protein–nucleic acid pair.

Reaction Conditions

Not directly applicable as a chemical reaction medium. For EMSA complex formation (general literature guidance; not product specifications):

• Temperature: On ice to room temperature; lower temperatures often stabilize weak complexes and improve resolution.
• Time: 10–30 min incubation is common; extend for slow binders.
• Ionic strength: Start low (e.g., 25–50 mM monovalent salt) and titrate upward to challenge specificity without abolishing binding.
• Cofactors: Include Mg2+ or Zn2+ only if required by the protein; avoid chelators when metal dependence is critical.
• Redox: Maintain reducing conditions (e.g., 0.05–1 mM DTT/TCEP) to preserve cysteine‑rich proteins.
• Additives: 5–10% glycerol aids loading/stability; trace nonionic detergent can reduce stickiness.

These are general EMSA binding conditions and must be optimized per target system. Item‑specific ranges are not specified for this SKU.

Safety and Handling

• GHS classification, pictograms, hazard statements: Not specified for this item; refer to SDS.
• Signal word: Not specified for this item; refer to SDS.
• General lab safety (good practice):
  • Wear appropriate PPE: lab coat, safety glasses, disposable nitrile gloves.
  • Avoid microbial contamination; keep containers closed. Use sterile technique if downstream assays require nuclease‑free conditions.
  • Prevent contact with eyes/skin; wash thoroughly after handling. Do not ingest or inhale aerosols/mists.
• Incompatibilities (general): Avoid mixing with strong oxidizers or acids/bases that could shift pH outside the intended range. Reducing agents (e.g., DTT) are air‑sensitive; minimize exposure to air and elevated temperature. Metal‑dependent formulations may be affected by chelators (e.g., EDTA) present in samples.
• First‑aid overview (general guidance; defer to SDS):
  • Eye/skin contact: Rinse with water for several minutes; remove contaminated clothing; seek medical attention if irritation persists.
  • Inhalation: Move to fresh air; seek attention if symptoms occur.
  • Ingestion: Rinse mouth; seek medical advice.
• Spill/cleanup: Wipe with absorbent material; wash area with water and mild detergent. Dispose according to institutional and local regulations.
• Research Use: For research use only. Not for human or veterinary use.
• SDS: Obtain the product‑specific SDS for authoritative hazard, exposure limits, and disposal guidance.

Solvent Selection

This product is an aqueous buffer system rather than a solvent to be selected; however, solvent compatibility matters for EMSA set‑ups.

• Polarity/miscibility: Water‑based, fully miscible with nuclease‑free water and compatible with standard native PAGE running buffers (e.g., TBE/TGE) when properly diluted.
• Additive compatibility (general literature guidance):
  • Low levels of glycerol (5–10%) aid complex stability and sample loading; higher percentages can increase viscosity and alter mobility.
  • Nonionic detergents (0.005–0.05%) can reduce nonspecific adsorption; excessive detergent can disrupt weak complexes.
  • Organic cosolvents (e.g., DMSO ≤2–5%) may be tolerated if ligands require solubilization, but can weaken protein–DNA interactions; titrate empirically.
• When to choose a dedicated EMSA binding buffer vs. DIY formulations:
  • Choose the dedicated buffer for speed, reproducibility, and QC. Opt for custom/DIY when a unique ionic composition, pH, or cofactor set is required for a particular protein.
• Comparison (general):
  • Dedicated EMSA buffer: ready‑to‑use, consistent, reduces variables.
  • DIY Tris/KCl/MgCl2 systems: maximal control over composition but increased batch‑to‑batch variability and preparation time.

Note: Item‑specific co‑solvent limits and pH range are not specified; verify compatibility with your gel system and targets.

Storage and Reconstitution

• Storage conditions (item‑specific): Store at −20 °C. Avoid repeated freeze–thaw cycles.
• Shipping (item‑specific): Shipped in an ice chest with ice pads to maintain the cold chain.
• Concentration/format: Not specified for this item; refer to CoA/Spec Sheet. If supplied as a concentrate (e.g., 5×/10×), thaw fully and mix gently before dilution.
• Handling and aliquoting (general best practice):
  • Thaw on ice or at 2–8 °C. Mix by gentle inversion; avoid vortexing if foaming could denature proteins or alter performance.
  • Aliquot upon first thaw to single‑use portions to minimize freeze–thaw cycles, especially if the buffer contains reducing agents.
  • Store working aliquots at −20 °C; short‑term storage at 2–8 °C may be acceptable for a few days depending on composition (not specified—verify on CoA).
• Stability/shelf life: Not specified for this item; refer to CoA/Spec Sheet. Monitor for precipitates, pH drift, or loss of performance as indicators of degradation.
• Reconstitution: If provided lyophilized or as a concentrate (not specified), reconstitute/dilute with nuclease‑free water to the indicated concentration per the product insert.

For definitive instructions, consult the product’s CoA/Spec Sheet and SDS.

Structure and Identity

• Item type: Formulated buffer for electrophoretic mobility shift assays (EMSA; gel-shift assays). Typically used to promote specific protein–DNA/RNA complex formation prior to native PAGE.
• Composition: Not specified for this item; refer to CoA/Spec Sheet. The product is a mixture rather than a single defined compound, so a single molecular formula or structure does not apply.
• CAS, CID, InChIKey, SMILES: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight / formula: Not applicable for a multi‑component buffer; per‑component identities are not provided in the Product Data.
• Structural features (general knowledge, literature): EMSA binding buffers commonly include a pH buffer (e.g., Tris or HEPES), monovalent salt (KCl or NaCl) to tune ionic strength, divalent cations (Mg2+) when required, a reducing agent (e.g., DTT or TCEP) to preserve protein activity, glycerol to increase sample density and stabilize complexes, nonionic detergent (e.g., NP‑40/Tween‑20) at trace levels to reduce nonspecific adsorption, and competitor nucleic acid (e.g., poly(dI·dC)) for specificity. These are typical components reported in the literature and are not specifications for this item.
• 2D description (general): As a buffer mixture, the “structure” is a solution containing small‑molecule buffering agents, salts, and stabilizers; no single ring system or discrete functional group profile can represent the product as a whole.

Synthetic Utility

Not typically applicable. This product is a biological assay buffer, not a synthetic reagent or building block.

• If chemical modification of nucleic acids or proteins is required, perform those steps separately and ensure residual reagents (e.g., DMSO, crosslinkers, chelators) are compatible with EMSA binding conditions.
• For in‑gel reactions (e.g., renaturation or on‑gel binding), verify that buffer components will not interfere with desired transformations.

Target Specificity

Target, epitope, species reactivity, clone, and isotype are applicable to antibodies or binding proteins, not to a buffer. For this product, such attributes are not applicable. Item‑specific target details are not specified for this item.