Pepsin Aqueous Solution (0.4%) - BioReagent,for microscopy,Suitable for Immunohistochemistry(IHC)

Cat. No.: G1508994

GRADE & PURITY BioReagent, for Microscopy, Suitable for Immunohistochemistry(IHC)

Synonyms

Pepsin Aqueous Solution

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Application

Cell Staining, IHC

Visão geral

  During tissue preparation, antigens are masked by the action of chemical reagents, and the peptide chains of some antigens are denatured by heat, making them undetectable during immunohistochemical staining. To address these issues, the process of re-exposing or restoring such antigens using chemical reagents and heat is referred to as antigen retrieval. Buffers such as citrate, EDTA or Tris can re-expose antigens masked by formalin under heating conditions without damaging antigenic epitopes, thereby improving antigen detection rates, reducing background staining and enhancing diagnostic accuracy.

  Prior to the advent of heat-induced antigen retrieval, enzyme-induced epitope retrieval using trypsin, Proteinase K, pepsin and other enzymes was the most commonly used method. 0.4% Pepsin Aqueous Solution can expose antigenic epitopes in samples such as paraffin sections, thus significantly improving immunostaining results. Paraffin sections generally require antigen retrieval treatment, while frozen sections do not necessarily need such treatment. Antigen retrieval can enhance the immunostaining effect of paraffin sections and also improve that of frozen sections to varying degrees; antigen retrieval may be considered when the immunostaining effect of frozen sections is unsatisfactory. Based on the calculation of 10 ml of 1× antigen retrieval solution required per slide, 100 ml of the solution can be used for the antigen retrieval of 10 samples.

Materials to Be Prepared by the User

1. Graded ethanol

2. Double-distilled water or deionized water

3. Heating equipment

4. Immunostaining wash buffer

Operating Procedures (For Reference Only)

(1) Paraffin Sections

1. Deparaffinization to water

① Xylene, 3 times, 3-5 minutes each time.

② Anhydrous ethanol for dehydration, 3 times, 3-5 minutes each time.

③ 95% ethanol, 3-5 minutes.

④ 90% ethanol, 3-5 minutes.

⑤ 80% ethanol, 3-5 minutes.

⑥ 70% ethanol, 3-5 minutes.

⑦ Rinse with distilled water, 2 times, 3-5 minutes each time.

2. Antigen retrieval: Immerse the sections in 0.4% Pepsin Aqueous Solution and incubate at 37 ℃ for 10-25 minutes.

3. Wash with immunostaining wash buffer or tap water, 1-2 times, 3-5 minutes each time, and rinse thoroughly.

4. Perform subsequent immunostaining steps.

(2) Frozen Sections

1. Wash the sections with immunostaining wash buffer or tap water for 5 minutes.

2. Antigen retrieval: Immerse the sections in 0.4% Pepsin Aqueous Solution and incubate at 37 ℃ for 10-25 minutes.

3. Wash with immunostaining wash buffer or tap water, 1-2 times, 3-5 minutes each time, and rinse thoroughly.

4. Perform subsequent immunostaining steps.

(3) Other Samples

For other samples, operate with reference to the procedures for paraffin sections or frozen sections.

Precautions

1. Allow the 0.4% Pepsin Aqueous Solution to return to room temperature before use, and avoid repeated freeze-thaw cycles.

2. The optimal incubation time for immersion in 0.4% Pepsin Aqueous Solution needs to be optimized by the user according to different samples and target proteins.

3. For your safety and health, wear a lab coat and disposable gloves during operation.

4. Use the reagent as soon as possible after opening to avoid compromising the results of subsequent experiments.

5. This product is for research use only and shall not be used for any other purposes.

Specifications

Sinónimos

Pepsin Aqueous Solution

Especificações e pureza

BioReagent,for microscopy,Suitable for Immunohistochemistry(IHC)

Estabilidade e armazenamento

Store at -20℃ long term (12 months).

Condições de armazenamento de armazenamento

Store at -20°C

Enviado em

Ice chest + Ice pads

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

Grau

BioReagent, for Microscopy, Suitable for Immunohistochemistry(IHC)

Product Line

Produtos biológicos

Application Protocols

The following are literature-based example protocols to assist method development with a 0.4% pepsin aqueous solution. Optimize for your tissues and antibodies. These are not validated specifications for this item.

A. IHC proteolytic antigen retrieval on FFPE sections (literature example)

• Deparaffinize and rehydrate sections through xylene and graded alcohols to water.
• Pre-equilibrate in cold acidic buffer (e.g., 0.01 N HCl or 0.1 M citrate, pH 2.0–2.5). Buffer composition for this item is not specified.
• Apply pepsin solution:
  • Starting point: 0.4% as supplied or dilute to 0.1–0.5% (1–5 mg/mL to 1–5 mg/mL equivalent) depending on tissue sensitivity.
  • Incubate 5–15 min at 25–37 °C in a humid chamber. Monitor under a microscope to avoid over-digestion.
• Stop reaction: Rinse thoroughly in cold PBS or Tris-buffered saline; proceed to blocking and primary antibody incubation.
• Notes: Adjust time/temperature/concentration per antigen. For fragile tissues, consider lower temperature or shorter times.

B. Preparation of F(ab')2 antibody fragments (research workflow, literature)

• Dissolve IgG in acidic buffer (e.g., 0.1 M citrate, pH ~3.5). Cool to 37 °C.
• Add pepsin to an enzyme:substrate ratio commonly ranging 1:100 to 1:1000 (w/w).
• Incubate 30–120 min at 37 °C with gentle mixing; sample periodically.
• Quench by raising pH to ~7–8 (e.g., with Tris) and/or adding pepstatin A.
• Purify F(ab')2 by protein A/G depletion of Fc remnants and SEC.

C. ISH permeabilization (literature)

• Brief exposure (e.g., 1–10 min at 37 °C) to dilute pepsin in acidic buffer to enhance probe penetration; verify that target nucleic acid integrity is preserved.

Always include appropriate controls. Document lot, time, temperature, pH, and concentration for reproducibility.

Biological Roles

General background (literature):

• Pepsin is a principal gastric aspartic protease (EC 3.4.23.1) formed from pepsinogen via autocatalytic activation in acidic conditions.
• Function: Initiates protein digestion in the stomach by cleaving peptide bonds preferentially adjacent to hydrophobic/aromatic residues (e.g., Phe, Tyr, Leu). Generates peptides that are further processed by pancreatic proteases.
• Structural/enzymatic features: Two-lobe structure with a catalytic dyad of Asp residues; operates optimally at low pH due to protonation states that support catalysis.
• Specificity profile: Favors cleavage at the N-terminal side of hydrophobic residues; avoids bonds involving basic residues when protonated environments prevail.
• Inhibition: Tightly inhibited by the pentapeptide inhibitor pepstatin A; activity is reduced at neutral pH and by denaturants.

Relevance to laboratory use:

• The low-pH activity enables selective proteolysis where neutral proteases (e.g., trypsin) are unsuitable.
• In histology/IHC, this biochemistry is exploited to unmask epitopes crosslinked by aldehyde fixatives, thereby enhancing antibody access without high-temperature retrieval.

Buffer Applications

This product is an enzyme solution rather than a buffering agent. It is not typically used to establish or maintain pH.

• For optimal performance, pepsin is generally employed in acidic buffers (e.g., dilute HCl, glycine-HCl, citrate) prepared separately and used to dilute the enzyme to working strength (literature).
• Select the buffer based on tissue compatibility, target antigen sensitivity, and downstream detection chemistry. See Reaction Conditions and Application Protocols for guidance.

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

Green Alternatives

Enzymatic antigen retrieval and tissue permeabilization can reduce reliance on harsher chemical or thermal methods.

Comparison (general, literature):

• Pepsin (this product):
  • Advantages: Biodegradable biocatalyst; effective at low temperatures and low concentrations; aqueous media; reduced energy input vs high-temperature HIER.
  • Tradeoffs: Acidic conditions required; potential for over-digestion and tissue damage if not optimized; protein origin may raise sourcing considerations.
• Proteinase K:
  • Advantages: Broad substrate tolerance; active over wider temperature/pH ranges.
  • Tradeoffs: Less selective; can rapidly over-digest delicate tissues; typically requires higher temperatures.
• Heat-Induced Epitope Retrieval (HIER):
  • Advantages: No added protease; avoids proteinaceous reagents.
  • Tradeoffs: High energy consumption (pressure cooker/microwave); potential for tissue detachment; buffer disposal considerations.

Greener practice recommendations:

• Use minimal effective enzyme concentration and shortest effective incubation time.
• Work at moderate temperatures (e.g., 25–37 °C) when compatible with the target.
• Prepare only required working volumes to minimize waste; avoid adding organic cosolvents.
• Dispose of proteinaceous waste through standard aqueous waste streams per local guidance.

Pharmaceutical Uses

No therapeutic or clinical claims are made for this product. For research use only.

General formulation/manufacturing context (literature):

• Pepsin is sometimes used as a processing aid in research and development settings for controlled proteolysis, including generation of F(ab')2 antibody fragments under acidic conditions, or removal of carrier proteins from conjugates. Such uses require validation of residuals and inactivation/removal steps in downstream processing.
• Excipient status/compendial listings for pepsin exist in some pharmacopeias for non-research materials; however, this specific item’s regulatory status, source, and suitability for GMP use are not specified.

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

If exploring process development applications, perform risk assessments on enzyme source, potential adventitious agents, and clearance, and confirm with supplier documentation.

Physical Properties

Item-specific physical specifications for this aqueous enzyme solution are not provided in the Product Data. Please consult the CoA/SDS for definitive values.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Concentration: 0.4% (w/v) pepsin in water (from Product Name).
• pH: Not specified for this item; refer to CoA/Spec Sheet.
• Density: Not specified for this item; refer to CoA/Spec Sheet.
• Osmolality/ionic strength: Not specified for this item; refer to CoA/Spec Sheet.
• Viscosity: Not specified for this item; refer to CoA/Spec Sheet.
• Solubility: Readily miscible with water by formulation; compatibility with organic solvents depends on denaturation/precipitation risk (general).
• UV–Vis characteristics: Not specified for this item; refer to CoA/Spec Sheet.

General (literature) properties of pepsin solutions:

• Optimal activity typically in strongly acidic media (approx. pH 1–4), with an activity maximum near pH ~1.5–3 (literature). This item’s formulation pH is not specified.
• Temperature activity profile commonly peaks near 37 °C and declines upon heating; irreversible denaturation occurs on prolonged exposure to elevated temperatures (literature).

Note: Do not treat literature values as product specifications. Use the provided CoA and method-specific validations for your application.

Quality and Grades

• Grade: BioReagent, for Microscopy, Suitable for Immunohistochemistry (IHC) (from Product Data).

What this means (general guidance):

• BioReagent: Manufactured for research applications with attention to biological performance parameters (e.g., enzymatic activity/consistency) rather than clinical use. Typically controlled for common contaminants relevant to lab workflows.
• For Microscopy: Intended for sample preparation steps in histology/cytology (e.g., proteolytic digestion to enhance stain penetration or antigen unmasking) with visual endpoint compatibility.
• Suitable for IHC: Appropriate for antigen retrieval/digestion in immunohistochemical staining workflows on tissue sections, including FFPE and frozen sections, subject to user optimization.

Item-specific notes:

• Concentration: 0.4% aqueous solution; ready for further dilution or direct use as dictated by your protocol.
• Stabilizers/preservatives: Not specified for this item; refer to CoA/Spec Sheet.
• Activity units and lot-to-lot control: Not specified for this item; refer to CoA/Spec Sheet.

Practical implications:

• Expect reduced background compatible with microscopy endpoints when used under optimized conditions.
• Validate digestion time and temperature on each tissue/antigen to avoid over-digestion.

Documentation: For method validation, request the current Certificate of Analysis (CoA) and SDS to confirm lot-specific parameters relevant to your assay.

Reaction and Applications

Focused on histology and IHC workflows where controlled proteolysis is beneficial.

Research/manufacturer applications (expanded):

• Microscopy and IHC: Proteolytic antigen retrieval on FFPE or frozen sections to unmask epitopes occluded by crosslinking, improving antibody binding and staining intensity. Also enhances probe penetration in in situ hybridization (ISH) when appropriate (general/literature).
• Tissue processing: Gentle digestion to improve section permeability before immunostaining or to modulate extracellular matrix components for morphology studies (literature).
• Protein mapping/biochemistry: Acid protease for selective peptide bond cleavage, complementary to trypsin-based workflows under acidic conditions (general).

Mechanistic notes (literature):

• Aspartic protease mechanism via two catalytic Asp residues activating a water molecule for nucleophilic attack on the scissile amide bond; prefers hydrophobic residues at P1/P1' positions.
• Optimal in strongly acidic environments; rapidly loses activity at neutral/basic pH.

Practical tips:

• Pre-equilibrate sections in the chosen acidic buffer before applying enzyme.
• Start with minimal exposure times (e.g., 5–10 min at 37 °C) and titrate upward to prevent over-digestion and epitope loss (general guidance).
• Immediately stop digestion by thorough rinsing in cold buffer and shifting to neutral pH or by brief fixation if required by the workflow.
• Validate on control tissue for each antibody/antigen pair. Document time–temperature–pH to ensure reproducibility.

Reaction Conditions

General, literature-based guidance for pepsin-catalyzed proteolysis. Treat these as starting points and optimize for your system. They are not product specifications.

• pH: Strongly acidic conditions are required; typical working pH ~1.5–3.5 depending on substrate sensitivity. Activity drops sharply above pH ~4–5.
• Temperature: 25–37 °C is common for controlled digestions. Higher temperatures accelerate digestion but risk denaturation/over-digestion.
• Enzyme loading: For tissue sections (IHC), working strengths often range from ~0.1 to 2 mg/mL, with exposure times of 5–30 min. This product is 0.4% w/v (4 mg/mL) and may be used as-supplied or further diluted depending on tissue/antigen tolerance (user optimization required).
• Time: Begin with the shortest feasible time (e.g., 5–10 min) and increase incrementally while monitoring morphology/epitope integrity.
• Quenching/stop: Rinse thoroughly with cold buffer; return to neutral pH. In inhibitor-based workflows, pepstatin A can be used to suppress residual activity (literature).
• Compatibility: Avoid organic solvents, strong detergents, chaotropes, oxidants, and basic buffers. Divalent cations have limited direct effect but buffer composition and ionic strength can modulate substrate accessibility.
• Controls: Include no-enzyme and heat-inactivated enzyme controls to benchmark background and morphology preservation.

Always adapt conditions to your tissue/antigen and detection chemistry.

Safety and Handling

Safety data for this specific item are not fully specified in the Product Data. Always consult the product SDS for authoritative guidance.

• GHS classification, pictograms, signal word, H-statements: Not specified for this item; refer to SDS.
• Likely hazards (general): Protein enzymes may cause allergic sensitization upon inhalation or skin exposure; avoid aerosol generation. Aqueous acidic formulations can be irritant depending on pH (literature/general).
• Recommended PPE: Laboratory coat, suitable gloves (e.g., nitrile), eye protection (safety glasses/face shield). Use a biosafety cabinet or chemical fume hood if aerosol formation or splashing is possible.
• Handling notes:
  • Avoid repeated freeze–thaw to preserve enzyme activity.
  • Prevent contamination; use sterile/clean tools and aliquot upon receipt if frequent use is anticipated.
  • Incompatibilities: Strong bases (neutralize/inactivate), oxidants, organic solvents/denaturants (e.g., ethanol, acetone, strong detergents, chaotropes) can reduce activity (general).
• First-aid overview (general):
  • Skin/eye contact: Rinse with water for several minutes; remove contact lenses; seek medical advice if irritation persists.
  • Inhalation: Move to fresh air; seek medical attention if symptoms develop.
  • Ingestion: Rinse mouth; do not induce vomiting; seek medical attention.

Spill/leak response (general): Absorb with inert material, avoid aerosolization, clean with water/detergent. Dispose per institutional and local regulations.

For research use only.

Solvent Selection

This product is supplied as a 0.4% aqueous solution and is designed for use in water-based buffers.

• Polarity/miscibility: Fully miscible with water by formulation. Not recommended for use in neat organic solvents; many organic solvents or strong denaturants precipitate or inactivate proteins (general).
• Compatible media (general, literature):
  • Acidic aqueous buffers typically support pepsin activity (approx. pH 1–4). Common choices include dilute HCl, glycine-HCl, or citrate buffers at low pH. The exact pH and buffer composition for this item are not specified.
  • Avoid high ionic strength or basic pH which can reduce activity.
• When to choose: Use this aqueous enzyme for proteolytic digestion during antigen retrieval in IHC, tissue permeabilization, partial de-crosslinking of FFPE sections, or selective proteolysis workflows where an acidic protease is desired.
• Alternatives: Trypsin (neutral/slightly basic optimum), proteinase K (broad specificity, higher temperature tolerance), or heat-induced epitope retrieval (HIER) for antigens sensitive to acid proteolysis (general comparison). Choose based on antigen stability, tissue integrity, and downstream detection sensitivity.

Note: Buffer composition and pH critically influence performance. Optimize conditions empirically for each sample type.

Storage and Reconstitution

• Storage conditions (item-specific): Store at -20 °C (from Product Data).
• Shipping (item-specific): Shipped in ice chest with ice pads (from Product Data).
• Format: Aqueous enzyme solution, 0.4% (w/v).

Handling and stability (general guidance):

• Aliquot upon first thaw to avoid repeated freeze–thaw cycles that can reduce activity.
• Thaw on ice; mix gently by inversion. Avoid vigorous vortexing or foaming.
• Short-term storage of in-use aliquots at 2–8 °C may be acceptable for the working day; assess activity empirically for your assay.
• Do not store in basic buffers; maintain acidic environment for activity assays and neutral pH for long-term stability only if validated. Item’s exact formulation is not specified.

Reconstitution: Not applicable; supplied as a ready-to-use aqueous solution. If dilution is required, use freshly prepared, filtered acidic buffer appropriate for your application.

Shelf life and stabilizers: Not specified for this item; refer to CoA/Spec Sheet.

For research use only.

Structure and Identity

A ready-to-use protease preparation for histology and IHC workflows. This product is an aqueous solution containing pepsin at 0.4% (w/v).

• SKU: G1508994
• Product name: Pepsin Aqueous Solution (0.4%)
• 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 for protein enzymes; not specified for this item.

Structural features (general, literature):

• Pepsin is an aspartic endopeptidase (EC 3.4.23.1) typically derived from gastric mucosa (literature).
• Single-chain globular protein with two aspartate residues in the catalytic dyad; characteristic bilobal fold with a substrate-binding cleft (literature).
• Active site recognizes hydrophobic residues (e.g., Phe, Leu, Tyr) on the non-prime side; prefers cleavage between hydrophobic amino acids (literature).

2D description in words (general):

• No small-molecule 2D formula; pepsin is a polypeptide enzyme. Functionally, it presents an acidic catalytic pocket formed by two Asp residues that coordinate water for peptide-bond hydrolysis under acidic conditions (literature).

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

Synthetic Utility

While not a traditional small-molecule reagent, pepsin serves as a biocatalyst for peptide bond hydrolysis under acidic conditions.

Utility in the lab (general, literature):

• Selective proteolysis: Cleaves at sites adjacent to hydrophobic residues, enabling mapping of protein domains or limited proteolysis studies distinct from trypsin/chymotrypsin digests.
• Antibody fragment preparation: Under controlled acidic conditions, pepsin can generate F(ab')2 fragments by cleaving below the hinge region, preserving antigen-binding while removing Fc (literature). Requires precise control of pH, temperature, and time to avoid over-digestion.
• Sample preparation: Partial digestion to improve accessibility of epitopes for IHC, ISH, or other affinity-based detection methods in fixed tissues.

Advantages and caveats:

• Operates in water at ambient/physiological temperatures; avoids harsh chemicals.
• Highly sensitive to pH; outside its active range, activity diminishes rapidly. Over-digestion can compromise structural integrity of targets.

Note: Optimize each application empirically; consider orthogonal verification (SDS-PAGE, MS peptide mapping, or control stains) to confirm extent of digestion.

Target Specificity

This listing is an enzyme reagent, not an antibody or affinity reagent. Therefore, target specificity fields such as antigen name, clone, isotype, and species reactivity are not applicable.

• Item-specific immunoreactivity data: Not applicable.
• For antibody-related information, refer to the primary antibody supplier and your validated IHC protocol.

Perguntas frequentes

What does for microscopy mean?

for microscopy indicates that the material has been processed and tested for specialty industrial processing. Specifications focus on the impurities that affect performance in that application — for example, metal content for electronics, particle size for batteries, refractive index for optics, or moisture content for moisture-sensitive processes.

What does BioReagent mean?

BioReagent indicates that the product has been processed and tested for biological and biochemical applications. Specifications may include endotoxin limits, microbial controls, sterility, low DNase/RNase/protease activity, and verified biological activity where relevant.

How should this product be stored?

Store at ?20 °C. Freezer storage is required to maintain the specified shelf life.

How is this product shipped?

This product ships in an insulated container with ice pads. 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.