HRP Blotting ECL

Cat. No.: H615636

Storage

Store at 2-8°C

Shipped In

Wet ice

Specifications

Condições de armazenamento de armazenamento

Store at 2-8°C

Enviado em

Wet ice

Este produto requer transporte de cadeia fria. Serviços terrestres e outros serviços econômicos não estão disponíveis.

Application Protocols

Item-specific validated protocols and recommended dilutions are not provided for H615636; refer to the CoA/Spec Sheet and any included Instructions for Use for authoritative guidance.

General guidance (literature, not item-specific) for Western blot with HRP/ECL:

• After transfer and blocking, incubate with primary antibody, then HRP-conjugated secondary; wash thoroughly in TBST.
• If the ECL is supplied in two parts, mix equal volumes immediately before use; cover membrane for 1–5 minutes with gentle agitation.
• Drain excess reagent without letting the membrane dry; image promptly using a CCD/CMOS imager or X-ray film with appropriate exposure times.
• For strong signals, reduce exposure or antibody concentrations to maintain linear quantitation.

These steps are provided as general background only and should not be construed as product-specific instructions.

Biological Roles

This product is a detection reagent system rather than a biological molecule; no endogenous biological role applies to the formulation itself.

Context (general, literature-based):

• Enzyme component in use: Horseradish peroxidase (HRP) is a plant heme enzyme used exogenously as a reporter; it is not part of human metabolic pathways. The reagent detects HRP activity conjugated to antibodies or other probes.
• Chemiluminescent substrate: Luminol and enhancers are synthetic small molecules without native metabolic roles in mammalian systems. Their function here is to emit light upon HRP-catalyzed oxidation.
• Reaction environment: Typical buffers (TBS/TBST) maintain protein structure and HRP activity during detection on membranes; the reaction is performed post-transfer, outside of living systems.

Implications for experimental biology:

• The reagent translates binding events (antigen–antibody interactions) into a luminescent signal, enabling sensitive quantification of target proteins after electrophoretic separation.
• Signal dynamics (initiation, peak, decay) depend on HRP turnover, substrate concentration, enhancer composition, and buffer conditions; optimizing these parameters improves band linearity for densitometry.

No medical or clinical claims are associated with this product. It is supplied strictly for research use only, as stated in the product data.

Buffer Applications

The product itself is not a buffer but is used in conjunction with standard immunoblotting buffers. No item-specific buffer recipes are provided for H615636; the following are general compatibility guidelines.

General buffer guidance (literature/general):

• Transfer and wash buffers: TBS or PBS are common; include 0.05–0.1% Tween 20 for washing steps to reduce non-specific binding. Excess detergent can suppress chemiluminescence.
• Blocking buffers: 5% non-fat dry milk in TBST for many targets; use 3–5% BSA in TBST for phospho-proteins or when milk interferes. Fish gelatin can reduce background in certain cases.
• HRP inhibitors to avoid: Sodium azide (even residual amounts from antibody storage) inhibits HRP. Dialyze or desalt antibodies containing azide prior to use, or select azide-free formulations.
• pH considerations: Luminol-based ECL is generally more efficient in mildly alkaline conditions; TBS/TBST at pH 7.4–8.0 is standard for blotting and compatible with most commercial ECL systems.
• Chelators and metals: High levels of metal ions (Cu, Fe) can catalyze peroxide decomposition; EDTA at typical buffer concentrations does not usually impair HRP and may help sequester trace metals.

Preparation tips (general):

• Use freshly prepared buffers or validated stock solutions; filter to remove particulates that could cause speckled background.
• Equilibrate membranes in the chosen buffer prior to ECL incubation to ensure uniform wetting.

For any product-specific buffer requirements or recommended formulations, consult the CoA/Spec Sheet.

Green Alternatives

While ECL blotting reagents are aqueous and used in small volumes, greener laboratory practices can still reduce environmental impact. No item-specific formulation details are provided for H615636; the following are general sustainability considerations for chemiluminescent HRP detection.

Strategies (general):

• Minimize waste: Scale incubation volumes to just cover the membrane (e.g., 0.05–0.1 mL/cm²) in reusable, dedicated trays to reduce reagent and plastic use.
• Reusability: Some protocols allow brief reuse of fresh ECL mix within a single session for replicate membranes if no significant signal depletion is observed; validate locally to avoid performance loss.
• Imaging choices: Prefer digital CCD/CMOS imagers over X-ray film to eliminate developer/fixer chemicals and silver-containing waste.
• Detergent management: Use the minimal effective TBST concentration to ease wastewater treatment.
• Alternative modalities:
  • Fluorescent detection avoids peroxides and can allow multiplexing, but requires specialized imagers and may involve organic solvent washes.
  • Near-infrared fluorescence increases signal stability and dynamic range without chemistries that generate reactive oxygen species.

Comparison (general, literature):

• HRP ECL: Aqueous, no heavy metals in typical use; generates minimal hazardous waste but contains oxidizers.
• Fluorescence: No oxidizers; dyes may have persistence concerns; higher instrument power use.

Institutional compliance:

• Segregate oxidizer-containing waste as per local rules; avoid drain disposal if prohibited.

For product-specific environmental attributes (e.g., preservative identity, recyclability of packaging), refer to the CoA/SDS and packaging information.

Pharmaceutical Uses

Not applicable. This product is a laboratory detection reagent for immunoblotting and is provided strictly for research use only.

General information (context, not item-specific):

• Chemiluminescent HRP substrates are utilized in bioanalytical laboratories for in-process control assays and QC of biologics, but they are not components of human or veterinary drug products.
• No pharmacopeial monographs are applicable to generic “ECL blotting reagents” as they are multi-component, performance-qualified formulations, not single-entity excipients.
• Use is confined to analytical laboratories (e.g., Western blot method development, release testing of recombinant proteins, or identity/impurity profiling) where method validation and reagent qualification are conducted under internal quality systems.

No clinical or therapeutic claims are made or implied for this catalog item.

Physical Properties

Item-specific physical constants are not provided for this formulated reagent.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Density, refractive index, pH, viscosity: Not specified for this item; refer to CoA/Spec Sheet.
• Boiling point, melting point, vapor pressure: Not applicable/relevant for aqueous working solutions; not specified for this item.
• Solubility: Aqueous formulation; ready-to-use with membranes (general expectation for ECL systems; literature/general information).
• Light sensitivity: High; protect from ambient and UV light to preserve chemiluminescent activity (general practice for luminol-based reagents; literature).

Literature/general reference values for ECL-type systems (not item-specific):

• Optimal emission maximum: ~425–455 nm (blue) from luminol chemiluminescence, influenced by enhancer and pH (literature).
• Operational pH range: HRP/luminol systems generally perform in mildly alkaline buffers; many formulations function well near pH 8–9 (literature).
• Working temperature: Ambient laboratory temperature; reaction kinetics increase with temperature but stability may decrease (literature guidance).

Practical notes (general):

• Avoid metal contamination (Fe, Cu) that can catalyze premature peroxide decomposition.
• Use clean, detergent-free trays; residual bleach or azide inhibits HRP.

For exact specifications (pH, component concentrations, and stability), consult the product’s CoA and SDS. Do not treat the above literature values as specifications for this catalog item.

Quality and Grades

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

Interpretation for ECL blotting reagents (general, not item-specific):

• Performance-grade ECL reagents are typically qualified for Western blot detection sensitivity (e.g., capable of detecting low femtogram–picogram levels of HRP-labeled analyte under defined conditions). Such claims, if applicable, would appear on the CoA or product datasheet.
• Lot-to-lot consistency is critical; reputable ECL products are QC’d for background signal on blank membranes, signal-to-noise ratio with standard HRP conjugates, and linear dynamic range across exposures.
• Stabilizers and preservatives (if present) extend shelf life at 2–8°C but can inhibit HRP at high concentrations. Precise identities/levels, if used in this product, are not specified here and should be verified on the CoA/SDS.
• UV absorbance specifications and trace metal limits are generally not relevant to end-use performance for blotting reagents and are not provided here.

What to expect from this item category:

• Ready-to-use or two-component mix designed for brief membrane incubation followed by chemiluminescent imaging.
• Optimized for low background on PVDF and nitrocellulose.

For this specific catalog number (H615636), consult the CoA/Spec Sheet for any stated sensitivity, dynamic range, and recommended exposure guidance. In the absence of item-specific claims, verify performance empirically with your assay controls.

Reaction and Applications

Primary application: chemiluminescent detection of HRP-labeled antibodies/probes on Western blot membranes.

Mechanistic backdrop (literature/general):

• HRP catalyzes the oxidation of luminol by hydrogen peroxide via radical intermediates, generating an excited 3-aminophthalate species that emits blue light (~425–455 nm) upon relaxation. Phenolic enhancers increase quantum yield and extend signal duration by facilitating radical propagation and efficient electron transfer.

Practical considerations (general best practices for ECL workflows):

• Blocking: Use a compatible blocker (e.g., 5% non-fat milk in TBST or BSA when probing phospho-epitopes). Residual biotin or peroxidase-like activity in blockers/membranes can raise background.
• Washes: Thorough TBST washes minimize non-specific binding; avoid azide in buffers (HRP inhibitor).
• Incubation: Mix equal volumes of the luminol/enhancer and peroxide solutions immediately before use if supplied as two parts; incubate membrane 1–5 minutes to ensure uniform wetting.
• Imaging: Capture promptly after incubation; multiple exposures (seconds to minutes) help bracket the linear range. For strong bands, reduce exposure or substrate concentration.
• Membrane choice: PVDF yields high binding capacity; pre-wet in methanol then equilibrate in buffer. Nitrocellulose offers low background but is more fragile.
• Troubleshooting: High background—improve washes, reduce antibody concentrations, verify blocker compatibility. Weak signal—confirm HRP activity, verify transfer efficiency, and ensure substrate freshness/light protection.

Item-specific test conditions and sensitivity claims are not provided for this product; consult the CoA/Spec Sheet for any recommended procedures unique to H615636.

Reaction Conditions

Item-specific directions for use are not provided here; refer to the CoA/Spec Sheet for any instructions unique to H615636. The following represents general literature guidance for HRP ECL detection on Western blots and is not a specification for this product.

General conditions (literature/general):

• Temperature: Ambient (20–25°C). Cooler temperatures slow kinetics but can extend signal duration.
• Mixing: If supplied as two components (luminol/enhancer and peroxide), combine 1:1 immediately before use. Use enough volume to fully wet the membrane (e.g., 0.05–0.1 mL/cm²).
• Incubation time: 1–5 minutes with gentle rocking for uniform coverage. Longer incubations rarely improve signal and can increase background.
• Imaging window: Begin acquisition immediately after draining excess reagent; capture multiple exposures (e.g., 10 s, 30 s, 2 min) to bracket linear range.
• Buffers: Final washes in TBST, then brief rinse in TBS (detergent-free) can enhance wetting and reduce bubbles before ECL application.
• Expected performance: With high-sensitivity ECL systems, picogram quantities of HRP-conjugated targets are detectable; actual sensitivity depends on antibody quality, transfer efficiency, membrane type, and imaging hardware.

Troubleshooting (general):

• Weak/rapidly fading signal: Verify HRP activity; use fresh substrate; avoid azide; ensure membranes remain moist.
• High background: Improve washes; optimize blocker; reduce primary/secondary concentrations; ensure clean trays.
• Speckling/streaks: Filter buffers; remove lint/particulates; avoid touching membrane with tools.

Always defer to the item’s official instructions if provided.

Safety and Handling

• GHS classification, signal word, hazard statements, and pictograms: Not specified for this item; refer to the product SDS for authoritative safety information.
• Storage conditions (item-specific): Store at 2–8°C. Protect from light. Shipped on wet ice.
• General hazards for HRP ECL-type reagents (literature/general):
  • May contain hydrogen peroxide at low concentration; oxidizer—avoid contact with reducing agents and transition metals.
  • May contain phenolic enhancers and amine derivatives (e.g., luminol analogs) that can be irritants. Avoid inhalation, ingestion, and skin/eye contact.
  • Light-sensitive; prolonged exposure to light can degrade activity and potentially form decomposition products.
• Incompatibilities (general): Strong reducers, oxidizable organics, heavy metal salts, and sodium azide (azide inactivates HRP and can form heavy metal azides in plumbing).
• PPE and engineering controls:
  • Wear lab coat, nitrile gloves, and safety glasses; handle in well-ventilated area.
  • Use light-protective practices (foil-wrapped bottles, subdued lighting) when opening/aliquoting.
• First aid (general guidance—defer to SDS):
  • Skin: Wash with soap and water; remove contaminated clothing.
  • Eyes: Rinse cautiously with water for several minutes; seek medical attention if irritation persists.
  • Inhalation: Move to fresh air; obtain medical advice if symptoms occur.
  • Ingestion: Rinse mouth; seek medical attention.
• Spill/cleanup:
  • Absorb small spills with inert material; avoid fuels for oxidation; collect for disposal according to institutional and local regulations.

Always consult the current SDS for this specific product prior to use.

Solvent Selection

Not typically applicable—this is a formulated, aqueous detection reagent intended for direct application to blotted membranes rather than as a solvent.

General compatibility notes for HRP ECL systems (literature/general):

• Polarity/matrix: Aqueous buffer; compatible with PVDF and nitrocellulose membranes after blocking and antibody incubation.
• Miscibility: Ready to use without dilution unless specified on the product datasheet; if dilution is recommended, deionized water or the supplied buffer is used.
• Buffer compatibility: TBS/TBST (pH ~7.4–8.0) is commonly used; PBS/PBST also works in many cases. Avoid sodium azide (HRP inhibitor). Excess detergents (e.g., >0.1% Tween 20) may reduce signal; typical TBST is 0.05–0.1%.

When to choose alternatives (general):

• For fluorescent imaging workflows, select fluorophore-conjugated secondaries and appropriate imaging buffers instead of ECL.
• For alkaline phosphatase (AP)-based detection, choose AP chemiluminescent substrates; AP systems require different buffer conditions (e.g., avoid phosphate in certain cases).

Mini comparison (general):

• ECL (HRP): High sensitivity, broad dynamic range, requires chemiluminescence imager or X-ray film.
• Fluorescence: Multiplexing capability, stable signals, requires fluorescence-capable imager.
• Colorimetric (TMB/DAB): Visual readout, lower sensitivity, permanent precipitate on membrane.

For this item, follow the solvent/buffer instructions on the CoA/Spec Sheet if provided.

Storage and Reconstitution

• Storage (item-specific): Store at 2–8°C. Protect from light. Shipped on wet ice.
• Reconstitution: Not specified for this item; refer to CoA/Spec Sheet. If supplied as concentrates or separate components, follow the item’s Instructions for Use for any dilution or mixing requirements.
• Handling (general best practice):
  • Equilibrate to room temperature before opening cold bottles to minimize condensation.
  • Work quickly under low light; dispense using clean, dedicated plasticware to avoid metal contamination.
  • Do not freeze unless explicitly allowed by the product datasheet; repeated freeze–thaw can degrade performance.
  • Consider aliquoting into light-protective microtubes to minimize repeated light/air exposure.
• Stability/shelf life: Not specified for this item; refer to CoA/Spec Sheet. Discard if discoloration, precipitate, or significant performance loss is observed during QC blots.
• Compatibility: Use with PVDF or nitrocellulose membranes. Avoid sodium azide in buffers or antibody stocks as it inhibits HRP.

For definitive storage duration and reconstitution details, consult the product’s CoA, SDS, and any included Instructions for Use.

Structure and Identity

This product is a ready-to-use HRP chemiluminescent blotting reagent system (commonly termed “ECL”) for detecting horseradish peroxidase-conjugated probes on membranes.

• Item-specific identifiers:

  • SKU: H615636
  • Product Name: HRP Blotting ECL
  • CAS: Not specified for this item; refer to CoA/Spec Sheet.
  • 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 to the mixture; not specified for this item.

• Composition/structure (general knowledge for HRP ECL systems, not item-specific):

  • Typically comprises an aqueous buffer containing a luminol-type chemiluminophore, a peroxidase enhancer (e.g., substituted phenols), and hydrogen peroxide in separate or combined components.
  • Functional features: HRP catalyzes luminol oxidation by H2O2, producing an excited 3-aminophthalate that emits blue light upon relaxation.

• Structural description in words (general, literature-based):

  • The active chemiluminophore is an anilide (luminol) bearing a diimide ring; enhancers are often phenolic/aromatic compounds that accelerate radical generation; the formulation is buffered to maintain the HRP turnover regime conducive to sustained emission.

• Use context:

  • Designed for membrane-based immunoassays (Western blot) with HRP-labeled antibodies or streptavidin-HRP.

Note: Because this is a formulated reagent mixture, discrete molecular identifiers are not provided and should be consulted in the product CoA/SDS where available.

Synthetic Utility

Not applicable. HRP Blotting ECL is a formulated detection reagent for immunoblotting and is not used as a synthetic building block or reaction solvent.

General note (context):

• While horseradish peroxidase can catalyze certain biocatalytic oxidations in synthetic chemistry, this product is not supplied or qualified for preparative catalysis and should not be repurposed for chemical synthesis.

Users seeking oxidative enzymatic transformations should consult dedicated biocatalyst products and corresponding substrate-grade hydrogen peroxide/buffer systems.

Target Specificity

Not applicable to this product type. HRP Blotting ECL is a chemiluminescent detection reagent and does not possess antigen/epitope specificity, clone identity, isotype, or species reactivity.

• Tested applications, antigen specificity, and clone/isotype metadata are relevant to antibodies or targeted probes, not to ECL substrates.
• For target-specific information, refer to the HRP-conjugated antibody or probe used in conjunction with this reagent.

Perguntas frequentes

How should this product be stored?

Store at 2–8 °C. Refrigerated storage is required to maintain the specified shelf life.

How is this product shipped?

This product ships chilled on wet ice. Unpack on arrival and transfer it to the storage condition stated above.

What documentation is provided?

Available product documentation, including Certificates of Analysis (COA), Safety Data Sheets (SDS), and specification sheets, is shown in the product document area. Document availability and access follow the current site policy.