SERPINB4 Antibody, HRP conjugated

What is SERPINB4 and why is it significant in research applications?

SERPINB4 (Serpin B4), also known as leupin, peptidase inhibitor 11 (PI-11), or squamous cell carcinoma antigen 2 (SCCA-2), is a member of the ovalbumin family of serine proteinase inhibitors. This protein functions primarily as a protease inhibitor that modulates host immune responses against tumor cells . SERPINB4 shares approximately 91-95% sequence identity with SERPINB3 at the amino acid level, with both proteins likely evolving from a common ancestral gene .

In research contexts, SERPINB4 has gained significance due to its observed upregulation in various inflammatory conditions including asthma, atopic dermatitis, psoriasis, and tuberculosis . The protein is predominantly expressed in epithelial cells, with higher expression noted in patients with chronic inflammatory conditions such as chronic rhinosinusitis with nasal polyps (CRSwNP) . As a protein implicated in inflammation regulation, SERPINB4 represents an important target for investigating pathological mechanisms and potential therapeutic interventions.

What are the key technical specifications of SERPINB4 Antibody, HRP conjugated products?

SERPINB4 Antibody, HRP conjugated products typically share several important technical characteristics that researchers should consider when selecting reagents:

These specifications provide essential information for experimental design and protocol optimization when working with these antibodies.

What are the optimal sample preparation methods for SERPINB4 detection in different tissue types?

Sample preparation methodologies vary depending on the tissue type and intended application. For SERPINB4 detection, researchers should consider the following approaches:

For epithelial cell samples, which are primary expression sites for SERPINB4 :

• For cultured cells: Harvest cells using appropriate buffers containing protease inhibitors to prevent protein degradation.

• For tissue sections: Use optimal cutting temperature (OCT) compound for fresh-frozen specimens or paraffin embedding for fixed tissues.

For immunofluorescence detection in tissue sections, as demonstrated in studies of nasal mucosa :

• Deparaffinize and rehydrate paraffin-embedded tissue sections

• Perform antigen retrieval using retrieval buffer (pH 6.0)

• Block with 10% goat serum

• Incubate overnight at 4°C with primary antibodies

• Incubate with fluorophore-conjugated secondary antibodies (if using non-conjugated primary)

• Mount slides with DAPI-containing mounting medium

For protein extraction for Western blot or ELISA:

• Homogenize tissues in RIPA buffer supplemented with protease inhibitors

• Centrifuge lysates to remove debris

• Quantify protein concentration using standard methods (BCA or Bradford assay)

• Normalize sample concentrations before analysis

Proper sample preparation is crucial for obtaining reliable and reproducible results when detecting SERPINB4 in experimental systems.

What is the optimal protocol for ELISA using SERPINB4 Antibody, HRP conjugated?

For optimal ELISA performance using SERPINB4 Antibody, HRP conjugated, researchers should follow this methodological approach:

• Coating Phase:

  • Coat 96-well plates with capture antibody or target protein in carbonate-bicarbonate buffer (pH 9.6)

  • Incubate overnight at 4°C

  • Wash 3-5 times with PBS containing 0.05% Tween-20 (PBST)

• Blocking Phase:

  • Block non-specific binding sites with 1-5% BSA or 5% non-fat dry milk in PBST

  • Incubate for 1-2 hours at room temperature

  • Wash 3-5 times with PBST

• Sample Addition:

  • Add samples and standards diluted in blocking buffer

  • Incubate for 1-2 hours at room temperature or overnight at 4°C

  • Wash 3-5 times with PBST

• Detection Phase:

  • Add SERPINB4 Antibody, HRP conjugated at the recommended dilution of 1:500-1:1000

  • Incubate for 1-2 hours at room temperature

  • Wash 5 times with PBST

• Development Phase:

  • Add TMB (3,3',5,5'-Tetramethylbenzidine) substrate

  • Incubate for 15-30 minutes in the dark at room temperature

  • Add stop solution (usually 2N H₂SO₄)

  • Read absorbance at 450nm with 620nm as reference wavelength

This protocol can be adjusted based on specific experimental requirements and target protein concentration in samples.

How can researchers validate the specificity of SERPINB4 Antibody in experimental systems?

Validating antibody specificity is critical for generating reliable research data. For SERPINB4 Antibody, researchers should implement multiple validation approaches:

• Positive and Negative Controls:

  • Use cell lines with known SERPINB4 expression (A431 cells, COLO 320 cells)

  • Include human saliva samples as positive controls

  • Use tissues/cells where SERPINB4 is not expressed as negative controls

• Knockdown/Knockout Validation:

  • Implement siRNA knockdown targeting SERPINB4 (such as sequences "CTTGTGAACGCAATCTATT" and "CTGCAACATATCATGTTGA")

  • Compare antibody reactivity between wild-type and knockdown samples

• Recombinant Protein Competition:

  • Pre-incubate antibody with excess recombinant SERPINB4 protein before application

  • Observe signal reduction in competition experiments

• Cross-Reactivity Assessment:

  • Test reactivity with closely related proteins, particularly SERPINB3 which shares ~92% amino acid identity

  • Consider using specific peptide regions for immunization to develop more specific antibodies

• Multiple Detection Methods:

  • Compare results across different techniques (ELISA, Western blot, immunofluorescence)

  • Confirm signal correlation across methodologies

Thorough validation ensures experimental findings accurately reflect SERPINB4 biology rather than non-specific interactions.

What troubleshooting approaches are recommended for weak or inconsistent signals with SERPINB4 Antibody?

When encountering weak or inconsistent signals with SERPINB4 Antibody, HRP conjugated, researchers should consider the following methodological troubleshooting approaches:

• Antibody Dilution Optimization:

  • Test a range of dilutions beyond the recommended 1:500-1:1000

  • Create a dilution series (1:250, 1:500, 1:1000, 1:2000) to identify optimal concentration

• Antigen Retrieval Enhancement:

  • For tissue sections, compare different antigen retrieval methods (heat-induced vs. enzymatic)

  • Optimize pH of retrieval buffer (test pH 6.0 vs. pH 9.0)

  • Extend retrieval time if needed

• Signal Amplification Strategies:

  • Implement biotin-streptavidin amplification systems

  • Consider tyramide signal amplification (TSA) for very low abundance targets

  • Increase substrate incubation time within the linear range of detection

• Sample Quality Assessment:

  • Verify protein integrity through total protein stains

  • Test fresh samples to rule out protein degradation

  • Increase sample concentration if target protein levels are low

• Blocking Optimization:

  • Test alternative blocking agents (BSA, casein, commercial blockers)

  • Extend blocking time to reduce background

• Incubation Conditions:

  • Compare room temperature vs. 4°C incubation

  • Extend primary antibody incubation time (overnight at 4°C)

• Protocol Modifications for Special Samples:

  • For samples with high protease activity, increase protease inhibitor concentration

  • For mucus-rich samples (like nasal polyps), include additional washing steps

Systematic troubleshooting using this methodological framework can help identify and resolve the specific issues affecting detection sensitivity.

How can SERPINB4 Antibody be used to investigate inflammatory pathways in epithelial cells?

SERPINB4 Antibody, HRP conjugated, offers valuable opportunities for investigating inflammatory pathways in epithelial cells, where SERPINB4 is predominantly expressed . Researchers can implement the following methodological approaches:

• Cytokine Stimulation Studies:

  • Treat cultured epithelial cells with inflammatory cytokines known to upregulate SERPINB4 expression (IL-4, IL-5, IL-6, IL-17a)

  • Use SERPINB4 Antibody to quantify protein upregulation via ELISA

  • Establish dose-response and time-course relationships

• Air-Liquid Interface (ALI) Culture Systems:

  • Establish ALI cultures of primary human epithelial cells (such as nasal epithelial cells)

  • Apply recombinant SERPINB4 (1 μg/mL) to cultures

  • Measure downstream effects on inflammatory mediators like CXCL8/IL-8

• Co-localization Studies:

  • Perform dual immunofluorescence staining using SERPINB4 Antibody alongside markers of inflammation

  • Quantify co-localization using digital image analysis

  • Correlate SERPINB4 expression with inflammatory cell infiltration

• Knockdown/Overexpression Approaches:

  • Implement siRNA knockdown of SERPINB4 in epithelial cells as described in literature

  • Alternatively, overexpress SERPINB4 using appropriate expression vectors

  • Monitor changes in inflammatory cytokine production using multiplexed assays

• Patient-Derived Sample Analysis:

  • Compare SERPINB4 levels between healthy controls and patients with inflammatory conditions

  • Correlate SERPINB4 expression with clinical parameters and inflammatory biomarkers

  • Stratify patients based on SERPINB4 expression patterns

These approaches can be integrated into comprehensive research strategies to elucidate SERPINB4's role in epithelial cell inflammation regulation.

How can researchers differentiate between SERPINB3 and SERPINB4 given their high sequence homology?

Differentiating between SERPINB3 and SERPINB4 presents a significant technical challenge due to their 92% amino acid sequence identity . Researchers can employ these methodological strategies for discrimination:

• Selective Antibody Epitopes:

  • Use antibodies raised against unique peptide sequences in non-conserved regions

  • Verify epitope specificity through sequence alignment analysis

  • Consider custom antibody development if commercial options lack specificity

• Isoform-Specific PCR:

  • Design primers targeting non-conserved regions between SERPINB3 and SERPINB4

  • Implement real-time PCR with high annealing temperature for specificity

  • Include melt curve analysis to verify amplicon specificity

• Mass Spectrometry Approaches:

  • Apply targeted proteomics focusing on unique peptides

  • Implement selected reaction monitoring (SRM) or parallel reaction monitoring (PRM)

  • Analyze tryptic digests for isoform-specific peptide signatures

• Recombinant Protein Standards:

  • Use purified recombinant SERPINB3 and SERPINB4 as standards

  • Compare antibody reactivity patterns

  • Create standard curves for quantitative discrimination

• Functional Discrimination:

  • Exploit differences in protease inhibition profiles between SERPINB3 and SERPINB4

  • Implement activity-based protein profiling

  • Develop functional assays based on differential protease targeting

• Immunodepletion Strategy:

  • Sequentially deplete samples using highly specific antibodies

  • Analyze remaining protein to determine isoform composition

  • Combine with Western blot or ELISA for quantification

Given the challenge of discrimination, researchers should implement multiple approaches and include appropriate controls to ensure reliable isoform identification.

What approaches can be used to quantitatively assess SERPINB4 expression in relation to inflammatory cytokines?

Quantitative assessment of SERPINB4 expression in relation to inflammatory cytokines requires rigorous methodological approaches:

• Multiplexed ELISA Systems:

  • Implement bead-based multiplexed assays measuring SERPINB4 alongside inflammatory cytokines

  • Calculate correlation coefficients between SERPINB4 and cytokines

  • Perform multivariate analysis to identify cytokine patterns associated with SERPINB4 expression

• qPCR with Normalization:

  • Design primers specifically for SERPINB4 (distinguishing from SERPINB3)

  • Normalize to stable reference genes validated for inflammatory conditions

  • Calculate fold changes using the 2^(-ΔΔCt) method

• Dose-Response and Time-Course Studies:

  • Treat cells with varying concentrations of cytokines (IL-4, IL-5, IL-6, IL-17a)

  • Collect samples at multiple time points (6h, 12h, 24h, 48h, 72h)

  • Generate mathematical models of SERPINB4 induction kinetics

• Digital Droplet PCR (ddPCR):

  • Implement absolute quantification without standard curves

  • Precisely measure copy numbers of SERPINB4 and inflammatory mediator mRNAs

  • Assess stoichiometric relationships

• Quantitative Proteomics:

  • Apply stable isotope labeling approaches (SILAC, TMT, iTRAQ)

  • Quantify SERPINB4 alongside inflammatory signaling proteins

  • Identify protein interaction networks and modifications

• Single-Cell Analysis:

  • Implement single-cell RNA sequencing or CyTOF

  • Correlate SERPINB4 expression with cytokine profiles at cellular level

  • Identify cell subpopulations with distinct SERPINB4/cytokine signatures

• In Situ Hybridization with Image Analysis:

  • Perform RNAscope or similar for SERPINB4 and cytokine mRNAs

  • Quantify signal intensity using digital image analysis

  • Generate spatial correlation maps

These quantitative approaches provide robust frameworks for understanding the relationship between SERPINB4 and inflammatory cytokines in research contexts.

What are the optimal storage conditions and stability considerations for SERPINB4 Antibody, HRP conjugated?

Proper storage and handling of SERPINB4 Antibody, HRP conjugated, is essential for maintaining reagent performance and experimental reproducibility:

Recommended Storage Conditions:

• Store at -20°C or -80°C for long-term preservation

• Avoid repeated freeze-thaw cycles that can denature the antibody and reduce activity

• For HRP-conjugated antibodies, protect from light to prevent photobleaching of the enzyme

Buffer Composition:
The antibody is typically supplied in buffer containing:

• 50% Glycerol (cryoprotectant)

• 0.01M PBS, pH 7.4 (physiological buffer)

• 0.03% Proclin 300 (preservative) or 0.02% sodium azide

Aliquoting Strategy:

• Upon receipt, prepare small working aliquots in sterile microcentrifuge tubes

• Use volumes appropriate for 2-3 experiments to minimize freeze-thaw cycles

• Label with antibody details, date, and dilution information

Stability Considerations:

• Working dilutions should be prepared fresh and used within 24 hours

• Monitor HRP activity over time using control experiments

• For long-term studies, include positive controls from the same antibody lot to track potential degradation

Quality Control Practices:

• Periodically test antibody performance using consistent positive controls

• Implement standardized ELISA protocols to monitor sensitivity trends

• Document lot numbers and performance characteristics for critical experiments

Shipping and Temporary Storage:

• If temporary storage at 4°C is necessary, limit to 1-2 weeks

• Return to -20°C for periods of non-use exceeding one week

• Ensure cold chain maintenance during shipping and transfer

Following these storage recommendations helps ensure optimal antibody performance throughout the research project lifecycle.

How can researchers confirm antibody functionality after extended storage?

Confirming antibody functionality after extended storage is a critical quality control measure. Researchers should implement these methodological approaches:

• Activity Comparison with Fresh Standard:

  • Perform parallel ELISA using both stored antibody and a fresh reference standard

  • Calculate relative activity as a percentage of the reference standard

  • Establish acceptance criteria (e.g., >80% relative activity)

• Dose-Response Testing:

  • Create serial dilutions of the antibody (1:250 to 1:2000)

  • Compare dose-response curves to historical data

  • Assess both sensitivity (detection threshold) and dynamic range

• Positive Control Panel Testing:

  • Maintain a frozen set of positive control samples with known SERPINB4 levels

  • Test stored antibody against this panel periodically

  • Monitor for shifts in signal intensity or pattern

• HRP Enzyme Activity Assessment:

  • Test HRP activity directly using standard substrates (TMB, ABTS, OPD)

  • Compare reaction kinetics with reference standards

  • Evaluate colorimetric development times

• Spectral Analysis:

  • Perform absorbance scan of HRP-conjugated antibody

  • Compare spectral characteristics to manufacturer specifications

  • Assess for shifts indicating potential degradation

• Western Blot Analysis:

  • If compatible with the antibody, perform Western blot on standard samples

  • Evaluate band intensity, specificity, and background levels

  • Compare signal-to-noise ratio with reference standards

• Documentation and Trending:

  • Maintain records of antibody performance over time

  • Track key parameters (sensitivity, specificity, background)

  • Establish clear criteria for antibody replacement

These methodological approaches provide a comprehensive framework for evaluating antibody functionality following extended storage, ensuring research data quality and reliability.

How should researchers interpret variable SERPINB4 expression levels across different experimental systems?

Interpreting variable SERPINB4 expression levels requires careful consideration of biological and technical factors. Researchers should follow these methodological approaches:

• Normalization Strategies:

  • For protein quantification, normalize to total protein content

  • For cell-specific analysis, normalize to epithelial cell markers

  • For tissue sections, consider area-based normalization using digital pathology

• Biological Context Consideration:

  • Inflammatory status significantly impacts SERPINB4 expression

  • Th2 cytokines (IL-4, IL-5) upregulate SERPINB4 expression

  • Account for disease state when comparing patient samples

• Reference Range Establishment:

  • Develop laboratory-specific reference ranges using healthy controls

  • Stratify reference ranges by tissue type and sample processing method

  • Apply statistical approaches to define "normal" variation

• Technical Variation Assessment:

  • Calculate intra-assay and inter-assay coefficients of variation

  • Implement quality control samples within each experimental run

  • Consider batch effects in longitudinal studies

• Correlation with Clinical Parameters:

  • For patient samples, correlate SERPINB4 levels with disease severity

  • Perform multivariate analysis incorporating clinical variables

  • Consider SERPINB4:SERPINB3 ratio as potentially more informative than absolute values

• Cell-Type Specific Analysis:

  • SERPINB4 is predominantly expressed in epithelial cells

  • Variations in epithelial cell content can significantly impact total SERPINB4 measurements

  • Consider single-cell approaches for heterogeneous samples

• Transcriptional vs. Protein-Level Regulation:

  • Compare mRNA and protein expression patterns

  • Investigate post-transcriptional regulation mechanisms

  • Consider protein stability and turnover rates

By systematically addressing these factors, researchers can develop more robust interpretations of SERPINB4 expression data across experimental systems and between patient cohorts.

What are the methodological considerations for using SERPINB4 Antibody in multiplex detection systems?

Incorporating SERPINB4 Antibody, HRP conjugated into multiplex detection systems requires specific methodological considerations:

• Cross-Reactivity Assessment:

  • Perform comprehensive cross-reactivity testing with all target proteins in the multiplex panel

  • Pay particular attention to SERPINB3 cross-reactivity due to high sequence homology

  • Validate specificity using knockout/knockdown controls

• Signal Separation Strategies:

  • When using multiple HRP-conjugated antibodies, implement sequential detection with HRP inactivation between steps

  • Consider alternative enzyme conjugates (AP, β-gal) for true multiplexing

  • Implement spectral unmixing algorithms for fluorescence-based multiplexing

• Dynamic Range Optimization:

  • Adjust antibody concentrations to accommodate different abundance levels of targets

  • Implement dual-scale detection systems for targets with widely varying concentrations

  • Verify linear response range for each target in the multiplex context

• Buffer Compatibility:

  • Test for compatibility between optimal buffers for SERPINB4 detection and other targets

  • Optimize buffer conditions to balance detection efficiency across all targets

  • Consider sequential incubation approaches if buffer requirements conflict

• Data Normalization Approaches:

  • Implement target-specific internal controls

  • Apply computational algorithms to normalize cross-assay variations

  • Consider spike-in controls for absolute quantification

• Spatial Multiplexing Considerations:

  • For tissue-based multiplex assays, optimize antigen retrieval conditions for all targets

  • Implement cyclic immunofluorescence or mass cytometry for high-parameter tissue analysis

  • Validate staining patterns using single-stain controls

• Validation Requirements:

  • Validate multiplex results against single-plex gold standards

  • Perform spike-recovery experiments to assess matrix effects

  • Establish limits of detection and quantification in multiplex context

These methodological considerations provide a framework for successfully integrating SERPINB4 Antibody, HRP conjugated into multiplex detection platforms while maintaining assay performance and data quality.

How is SERPINB4 research contributing to our understanding of epithelial inflammation mechanisms?

SERPINB4 research has revealed important insights into epithelial inflammation mechanisms, with several methodological approaches driving new discoveries:

• Transcriptomic Analysis:
  Studies have identified differentially expressed genes in response to recombinant SERPINB4 stimulation, with pathway enrichment analysis showing that downregulated genes are enriched in cytokine-cytokine receptor interactions . This suggests SERPINB4 may function as a negative regulator of specific inflammatory pathways.

• Cytokine-Mediated Regulation:
  Research demonstrates that SERPINB4 expression is upregulated by multiple inflammatory cytokines including IL-4, IL-5, IL-6, and IL-17a , suggesting this protein responds to diverse inflammatory signals and potentially acts as a feedback regulator.

• Disease Association Studies:
  Increased expression of SERPINB4 has been found in multiple inflammatory conditions including asthma, atopic dermatitis, psoriasis, and tuberculosis . In chronic rhinosinusitis with nasal polyps (CRSwNP), SERPINB4 expression is elevated in both eosinophilic and non-eosinophilic forms of the disease .

• Protein-Protein Interaction Networks:
  Network analysis of SERPINB4-responsive genes has identified CXCL8 (IL-8) as a hub gene , suggesting SERPINB4 may modulate neutrophil recruitment and activation in inflammatory microenvironments.

• Mechanistic Investigations:
  Experimental approaches using air-liquid interface cultures of primary human nasal epithelial cells have shown that recombinant SERPINB4 treatment affects CXCL8/IL-8 expression , providing direct evidence for its role in modulating epithelial inflammatory responses.

• Genetic Association Studies:
  Both SERPINB3 and SERPINB4 have been associated with disease genes of nasal polyps and inflammation in the DisGeNET database , suggesting genetic variation in these genes may influence susceptibility to inflammatory conditions.

These research approaches collectively demonstrate that SERPINB4 is not merely a biomarker of inflammation but an active participant in regulatory networks controlling epithelial inflammatory responses.

What methodological advances are improving the specificity and sensitivity of SERPINB4 detection?

Methodological advances are continuously enhancing the specificity and sensitivity of SERPINB4 detection, with several key innovations:

• Improved Immunogen Design:
  Modern SERPINB4 antibodies utilize recombinant proteins covering specific amino acid regions (such as 128-208AA) that maximize unique epitopes while minimizing cross-reactivity with SERPINB3.

• Affinity Purification Techniques:
  Advanced antibody production now employs multi-step purification processes, including protein G purification and antigen-specific affinity chromatography, resulting in higher specificity preparations.

• Signal Amplification Systems:
  Beyond standard HRP conjugation, newer detection systems incorporate:

  • Polymer-based signal enhancement technologies

  • Tyramide signal amplification for ultra-sensitive detection

  • Quantum dot conjugation for improved stability and brightness

• Multiplex Capabilities:
  Technological advancements now enable simultaneous detection of SERPINB4 alongside related proteins and cytokines through:

  • Multiplexed immunoassay platforms

  • Multicolor flow cytometry with spectral unmixing

  • Sequential immunofluorescence techniques

• Digital Analysis Methods:
  Computational approaches improve detection accuracy through:

  • Automated image analysis for quantitative immunohistochemistry

  • Machine learning algorithms for pattern recognition

  • Digital pathology tools for tissue-based quantification

• Single-Cell Technologies:
  Emerging methods enable SERPINB4 detection at single-cell resolution:

  • Single-cell proteomics using mass cytometry (CyTOF)

  • Spatial transcriptomics for tissue-based localization

  • Imaging mass cytometry for multiplexed protein detection

• Validation Protocols:
  Improved validation approaches enhance confidence in results:

  • CRISPR/Cas9 knockout controls

  • siRNA knockdown with specific sequences targeting SERPINB4

  • Recombinant protein competition assays

These methodological advances collectively improve the reliability and utility of SERPINB4 detection in both basic research and clinical investigation contexts.