SERPINB4 Antibody, FITC conjugated

What is SERPINB4 and what is its biological function?

SERPINB4, also known as squamous cell carcinoma antigen 2 (SCCA-2), belongs to the ovalbumin-serine proteinase inhibitor (ova-serpin) family. It functions as a serine protease inhibitor that specifically targets cathepsin G and mast cell chymase, as well as the cysteine protease Derp1 found in house dust mite . SERPINB4 serves as a potent inhibitor of apoptosis and may modulate the host immune response against tumor cells . Recent research has demonstrated that SERPINB4 is expressed in human Th2 cells after in vitro polarization and in memory Th2 cells from pollen allergy patients, suggesting a role in allergic inflammation .

Methodologically, when studying SERPINB4's biological function, researchers should consider its relationship with its close homolog SERPINB3, as they share over 92% amino acid identity but differ in their reactive center loop sequences, which determines substrate specificity .

How does SERPINB4 differ structurally and functionally from SERPINB3?

While SERPINB3 and SERPINB4 share over 92% amino acid identity, they have only 54% identity in their protease-binding reactive center loop, which provides substrate specificity . This structural difference translates to functional distinctions:

• SERPINB3 (SCCA-1) inhibits both serine proteinases such as chymotrypsin and cysteine proteinases including cathepsins L, K, S, and papain

• SERPINB4 (SCCA-2) specifically inhibits serine proteinases such as cathepsin G and mast cell chymase , as well as the cysteine protease Derp1 from house dust mite

Both proteins function as potent inhibitors of apoptosis and may have overlapping roles in modulating immune responses . When designing experiments to distinguish between these closely related proteins, researchers should employ highly specific antibodies or genetic approaches that can discriminate between the two serpins.

What is the significance of SERPINB4 in allergic inflammation?

SERPINB4 has emerged as a significant factor in allergic inflammation, particularly in Th2-mediated responses. Studies have demonstrated that:

• Both SERPINB3 and SERPINB4 are expressed in human Th2 cells after in vitro polarization

• They are found in memory Th2 cells isolated from pollen allergy patients

• SERPINB4 can inhibit the cysteine protease Derp1 found in house dust mite, a common allergen

• The murine homolog Serpinb3a plays a non-redundant role in the induction of mucus production in asthma models

To investigate SERPINB4's role in allergic inflammation, researchers should consider experimental designs that include gene knockdown approaches as demonstrated in previous studies where shRNAmir targeting SERPINB3 or SERPINB4 was utilized to assess their function in CD4+CD27- T cells .

What are the key specifications of SERPINB4 Antibody, FITC conjugated?

The SERPINB4 Antibody, FITC conjugated (CSB-PA021072LC01HU) has the following specifications:

The FITC conjugation enables direct fluorescent detection in applications such as flow cytometry and immunofluorescence microscopy without the need for secondary antibodies .

What are the recommended applications for SERPINB4 Antibody, FITC conjugated?

Based on the antibody specifications and research on similar anti-SERPINB3/B4 antibodies, the SERPINB4 Antibody, FITC conjugated is suitable for the following applications:

• Flow Cytometry: The FITC conjugation makes it ideal for direct detection of SERPINB4 in cells without requiring secondary antibodies

• Immunofluorescence Microscopy: For visualization of SERPINB4 expression patterns in cells and tissues

• Confocal Microscopy: For high-resolution imaging of SERPINB4 subcellular localization

While not directly specified for the FITC-conjugated version, related antibodies against SERPINB3/B4 have been validated for:

• Western Blotting: Detecting SERPINB4 in cell or tissue lysates

• Immunohistochemistry: For paraffin-embedded tissues (IHC-P)

When using this antibody for new applications, researchers should perform appropriate validation studies including positive and negative controls to ensure specificity and optimal performance.

How should researchers optimize storage conditions for SERPINB4 Antibody, FITC conjugated?

To maintain optimal activity of the SERPINB4 Antibody, FITC conjugated:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles, as these can compromise antibody integrity and fluorophore activity

• For working solutions, aliquot and store at 4°C protected from light (FITC is light-sensitive)

• Consider adding sodium azide (0.02%) to aliquots unless used for applications where azide would interfere

• Monitor storage buffer conditions (pH 7.4) as extreme pH can affect antibody stability

For long-term storage stability assessment, researchers should periodically test antibody activity using positive control samples. FITC fluorescence can diminish over time, particularly if exposed to light or subjected to multiple freeze-thaw cycles, so proper handling is essential to maintain signal integrity.

How should researchers design experiments to investigate SERPINB4 in Th2-mediated allergic responses?

When designing experiments to investigate SERPINB4 in Th2-mediated allergic responses, researchers should consider the following methodological approach:

• Cell Isolation and Culture:

  • Isolate naïve CD4+CD45RA+ T cells from human peripheral blood mononuclear cells (PBMCs) using negative selection methods

  • Confirm purity (>95%) using flow cytometry with PE-conjugated anti-CD45RA antibodies

  • Culture cells in appropriate media (RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin)

• Th2 Polarization:

  • Stimulate cells with anti-CD3 (0.5μg/mL) and anti-CD28 (0.5μg/ml)

  • Add cytokines to induce Th2 polarization (e.g., IL-4) and block non-Th2 differentiation

• SERPINB4 Expression Analysis:

  • Measure SERPINB4 mRNA expression using quantitative PCR

  • Assess protein expression using the FITC-conjugated SERPINB4 antibody via flow cytometry

  • Compare expression in non-allergic controls versus cells from allergic patients

• Functional Studies:

  • Use gene knockdown approaches with shRNA targeting SERPINB4

  • Assess effects on cell viability, apoptosis, and cytokine production

  • Evaluate the impact on Th2 cell persistence and function

• In vivo Relevance:

  • Consider allergic mouse models (e.g., house dust mite or ovalbumin challenge)

  • Analyze the murine homolog Serpinb3a role in airway inflammation

This experimental design allows for comprehensive investigation of SERPINB4's role in allergic inflammation, from molecular expression to functional significance.

What controls should be included when using SERPINB4 Antibody, FITC conjugated in flow cytometry?

For robust flow cytometry experiments using SERPINB4 Antibody, FITC conjugated, researchers should include:

• Positive Controls:

  • Cell lines known to express SERPINB4 (e.g., squamous cell carcinoma lines)

  • HEK-293T cells transfected with SERPINB4 expression vector

  • Th2 polarized cells from allergic patients

• Negative Controls:

  • Isotype control: FITC-conjugated rabbit IgG at the same concentration

  • Cells known not to express SERPINB4 (e.g., certain non-squamous epithelial cells)

  • Adjacent normal tissue to carcinoma samples

  • HEK-293T cells transfected with empty vector

• Technical Controls:

  • Unstained cells to establish autofluorescence

  • Single-color controls for compensation if performing multicolor flow cytometry

  • FMO (Fluorescence Minus One) controls when performing multiparameter analysis

• Validation Controls:

  • Parallel analysis with unconjugated anti-SERPINB4 followed by FITC-conjugated secondary antibody

  • Pre-absorption of the antibody with recombinant SERPINB4 protein to confirm specificity

  • SERPINB4 knockdown cells to validate antibody specificity

Including these controls allows proper interpretation of results, ensures specificity, and helps troubleshoot potential technical issues.

What are the considerations for using SERPINB4 Antibody, FITC conjugated in immunofluorescence studies?

When using SERPINB4 Antibody, FITC conjugated for immunofluorescence studies, researchers should consider:

• Sample Preparation:

  • For fixed cells: Use 4% paraformaldehyde fixation followed by permeabilization with 0.1-0.5% Triton X-100

  • For tissue sections: Consider antigen retrieval methods if needed

  • Blocking step: Use 5-10% normal serum from a species different from the antibody host to reduce background

• Antibody Dilution and Incubation:

  • Start with manufacturer's recommended dilution (typically 1:100 to 1:500)

  • Perform a dilution series to determine optimal concentration

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

  • Protect from light during and after incubation due to FITC photosensitivity

• Counterstaining and Mounting:

  • Nuclear counterstain: DAPI or Hoechst (blue) works well with FITC (green)

  • Consider phalloidin-rhodamine for cytoskeletal visualization (red)

  • Use anti-fade mounting media to preserve FITC signal

• Controls and Validation:

  • Include positive control tissues known to express SERPINB4 (e.g., squamous cell carcinomas)

  • Include negative controls (normal pancreas shows minimal SERPINB4 expression)

  • Use peptide competition assay to confirm specificity

• Imaging Considerations:

  • FITC excitation maximum: ~495 nm; emission maximum: ~519 nm

  • Minimize exposure time to reduce photobleaching

  • Consider acquiring images quickly after preparation

  • If stored, keep slides in the dark at 4°C

Following these methodological guidelines ensures optimal signal-to-noise ratio and specificity in immunofluorescence studies using FITC-conjugated SERPINB4 antibody.

How can researchers investigate the mechanism of SERPINB4's anti-apoptotic function?

To investigate SERPINB4's anti-apoptotic function, researchers should employ a multifaceted approach:

• Molecular Interaction Studies:

  • Identify binding partners using co-immunoprecipitation followed by mass spectrometry

  • Validate interactions with purified proteins using surface plasmon resonance or ELISA

  • Focus on known apoptotic pathway components, particularly those related to JNK1 signaling

• Cellular Assays:

  • Compare apoptosis rates in cells with normal versus knocked-down SERPINB4 expression

  • Use flow cytometry with Annexin V/PI staining to quantify apoptotic populations

  • Apply specific apoptotic stimuli (UV radiation, FasL, cytotoxic drugs) to test pathway specificity

  • Monitor activation of caspases (especially caspase-3, -8, and -9) using fluorogenic substrates

• Mechanistic Investigation:

  • Assess SERPINB4's impact on JNK1 activity using phospho-specific antibodies and kinase assays

  • Investigate how SERPINB4 inhibits its protease targets (cathepsin G, mast cell chymase)

  • Evaluate subcellular localization during apoptosis using the FITC-conjugated antibody

  • Create domain-specific mutants to map regions responsible for anti-apoptotic function

• In vivo Relevance:

  • Use animal models with modified expression of the murine homolog (Serpinb3a)

  • Correlate SERPINB4 expression with apoptosis markers in human tissue samples

  • Analyze tissues from malignancies where SERPINB4 is overexpressed

This comprehensive approach will help elucidate whether SERPINB4's anti-apoptotic function stems from direct protease inhibition or involves additional non-canonical mechanisms.

What approaches can researchers use to distinguish between SERPINB3 and SERPINB4 in experimental systems?

Distinguishing between SERPINB3 and SERPINB4 presents a significant challenge due to their 92% amino acid identity . Researchers can employ these strategies:

• Genomic and Transcriptomic Approaches:

  • Design PCR primers targeting the divergent reactive center loop region (54% identity)

  • Use RNA-Seq with computational algorithms to distinguish between isoforms

  • Employ droplet digital PCR for absolute quantification of each transcript

  • Design specific siRNAs targeting unique regions for selective knockdown

• Proteomic Methods:

  • Develop antibodies against the reactive center loop unique to each protein

  • Use mass spectrometry to identify peptides specific to each protein

  • Employ 2D gel electrophoresis to separate proteins based on slight differences in pI

• Functional Discrimination:

  • Test inhibition against different proteases:

    • SERPINB3-specific targets: cathepsins L, K, S

    • SERPINB4-specific targets: cathepsin G, mast cell chymase

  • Assess differential inhibition kinetics with purified proteins

  • Create chimeric proteins swapping the reactive center loops to confirm specificity determinants

• Genetic Manipulation:

  • Use CRISPR/Cas9 to specifically knock out either SERPINB3 or SERPINB4

  • Employ lentiviral shRNA constructs specific to each target

  • Generate expression constructs with epitope tags for differential detection

This methodological toolbox enables researchers to distinguish between these closely related serpins despite their high sequence similarity.

How can researchers investigate SERPINB4's role in tumor biology using FITC-conjugated antibodies?

To investigate SERPINB4's role in tumor biology using FITC-conjugated antibodies, researchers should implement a comprehensive strategy:

• Expression Profiling in Tumor Tissues:

  • Perform immunofluorescence microscopy on tissue microarrays containing various tumor types

  • Compare SERPINB4 expression between tumor tissue and adjacent normal tissue

  • Correlate expression with clinical parameters (stage, grade, patient outcome)

  • Create quantitative expression maps using digital image analysis

• Cellular Localization Studies:

  • Use confocal microscopy with FITC-conjugated SERPINB4 antibody to determine subcellular localization

  • Perform co-localization studies with markers for different cellular compartments

  • Compare localization patterns between normal and malignant cells

  • Track dynamic changes in localization during cell cycle or in response to stress

• Flow Cytometry Applications:

  • Isolate cells from primary tumors and analyze SERPINB4 expression

  • Correlate SERPINB4 levels with other markers of proliferation or stemness

  • Sort SERPINB4-high versus SERPINB4-low populations for functional studies

  • Assess changes in expression following treatment with chemotherapeutic agents

• Functional Studies in Tumor Models:

  • Generate SERPINB4-overexpressing and knockdown tumor cell lines

  • Assess impact on proliferation, migration, invasion, and resistance to apoptosis

  • Perform xenograft studies comparing growth and metastatic potential

  • Use intravital imaging with FITC-labeled antibodies to track SERPINB4-expressing cells in vivo

This approach leverages the advantages of FITC-conjugated antibodies, particularly for direct visualization applications, to comprehensively investigate SERPINB4's role in cancer biology, as suggested by its elevated expression in squamous cell carcinomas and pancreatic carcinoma .

What are common challenges in detecting intracellular SERPINB4 and how can they be addressed?

Detecting intracellular SERPINB4 presents several challenges that researchers can address with specific methodological approaches:

• Fixation and Permeabilization Issues:

  • Challenge: Inadequate cell permeabilization leading to poor antibody access

  • Solution: Optimize permeabilization conditions (test different detergents like saponin, Triton X-100, or digitonin at various concentrations)

  • Approach: For flow cytometry, compare commercial fixation/permeabilization kits to identify optimal conditions

• Cross-Reactivity Concerns:

  • Challenge: Difficulty distinguishing SERPINB4 from SERPINB3 due to 92% sequence homology

  • Solution: Use competitive binding assays with recombinant proteins

  • Approach: Validate specificity in systems with differential expression of SERPINB3 versus SERPINB4

• Low Expression Levels:

  • Challenge: Insufficient signal-to-noise ratio in cells with low expression

  • Solution: Implement signal amplification methods like tyramide signal amplification

  • Approach: Use positive controls like squamous cell carcinoma samples with known high expression

• Autofluorescence Interference:

  • Challenge: Cellular autofluorescence in the FITC channel

  • Solution: Include unstained controls and consider autofluorescence quenching reagents

  • Approach: Adjust instrument settings based on unstained control samples

• Fixation-Induced Fluorescence Loss:

  • Challenge: FITC signal degradation during fixation procedures

  • Solution: Minimize fixation time and protect from light

  • Approach: Consider alternative fixatives or post-fixation antibody staining

By systematically addressing these technical challenges, researchers can generate more reliable and reproducible data when studying intracellular SERPINB4 localization and expression.

How should researchers interpret discrepancies between SERPINB4 mRNA and protein expression data?

When faced with discrepancies between SERPINB4 mRNA and protein expression levels, researchers should consider these methodological approaches to interpretation:

• Potential Biological Explanations:

  • Post-transcriptional regulation: Assess miRNA targeting of SERPINB4 mRNA using prediction algorithms and validation studies

  • Protein stability differences: Measure protein half-life using cycloheximide chase experiments

  • Stimulus-specific regulation: Compare resting versus activated states (particularly relevant in immune cells)

  • Spatial-temporal variations: Consider differences in sampling time points and microenvironmental factors

• Technical Considerations:

  • Primer specificity: Verify that qPCR primers discriminate between SERPINB3 and SERPINB4

  • Antibody cross-reactivity: Validate antibody specificity against both SERPINB3 and SERPINB4 recombinant proteins

  • Detection sensitivity: Compare sensitivity thresholds of PCR versus immunological methods

  • Sample preparation differences: Standardize protocols for both RNA and protein extraction

• Experimental Validation Approaches:

  • Correlation analysis: Perform large-scale assessment across multiple samples to identify consistent patterns

  • Single-cell analysis: Use single-cell RNA-seq coupled with flow cytometry to assess correlation at individual cell level

  • Time-course studies: Track both mRNA and protein levels following stimulation

  • Translational efficiency: Assess polysome association of SERPINB4 mRNA

• Integrated Analysis Framework:

  • Create a mathematical model accounting for transcription rates, mRNA stability, translation efficiency, and protein degradation

  • Perform pulse-chase experiments to determine actual rates in your experimental system

  • Consider that certain cellular states may prioritize post-transcriptional regulation

This systematic approach helps researchers determine whether discrepancies represent technical artifacts or biologically meaningful regulatory mechanisms affecting SERPINB4 expression.

What factors should researchers consider when comparing SERPINB4 expression across different tissue types?

When comparing SERPINB4 expression across different tissue types, researchers should consider these methodological factors:

• Tissue-Specific Expression Patterns:

  • SERPINB4 shows preferential expression in squamous epithelium and certain carcinomas

  • Consider baseline expression levels in normal tissue counterparts (e.g., pancreatic carcinoma shows positive staining while adjacent normal pancreas does not)

  • Account for cellular heterogeneity within tissues using single-cell approaches or microdissection

• Standardization and Quantification Approaches:

  • Normalization strategy: Use multiple housekeeping genes/proteins suited to each tissue type

  • Quantification method: Establish consistent scoring systems for immunohistochemistry/immunofluorescence

  • Analytical controls: Include the same positive control sample across all experiments

  • Technical parameters: Standardize antibody concentration, incubation time, and detection systems

• Confounding Variables:

  • Fixation differences: Compare only tissues with similar fixation protocols

  • Autofluorescence: Especially problematic in certain tissues (e.g., liver, brain)

  • Sample handling: Account for ischemic time affecting protein preservation

  • Pathological state: Distinguish between normal, inflammatory, and neoplastic conditions

• Validation Strategies:

  • Confirm findings using orthogonal detection methods (e.g., Western blot, mass spectrometry)

  • Compare antibody-based detection with mRNA expression data from tissues

  • Use multiple antibodies targeting different epitopes of SERPINB4

  • Include tissues known to be negative for SERPINB4 as controls

This comprehensive approach ensures that observed differences in SERPINB4 expression between tissue types represent true biological variation rather than methodological artifacts, particularly important when comparing results from pancreatic, lung, kidney, and bladder tissues as shown in previous studies .