SERPINB2 Antibody, HRP conjugated

What is SERPINB2 and what are its functional characteristics?

SERPINB2 (Serpin Family B Member 2), also known as Plasminogen Activator Inhibitor-2 (PAI-2), is a 415 amino acid serine protease inhibitor belonging to the Serpin family, Ov-serpin subfamily . It primarily functions as an inhibitor of urokinase-type plasminogen activator . SERPINB2 shows high expression in blood, vasculature, and placenta, with notable expression also in skin and esophagus .

The protein is significantly upregulated during pregnancy and in activated monocytes/macrophages in response to various viral, bacterial, and parasitic agents . SERPINB2 can represent up to 1% of total protein in activated macrophages, making it one of the most upregulated proteins in these cells during inflammation .

Recent research has revealed additional physiological functions beyond protease inhibition, including:

• Regulation of Th1 immune responses

• Modulation of inflammatory processes

• Potential tumor suppressor activity in certain cancers

• Involvement in neurite outgrowth during post-natal brain development through interactions with CHL1 and Vitronectin

What are the key characteristics of HRP-conjugated SERPINB2 antibodies?

HRP-conjugated SERPINB2 antibodies combine target specificity with direct enzymatic detection capabilities. These reagents have several distinguishing features:

The primary methodological advantage of HRP-conjugated antibodies is the elimination of secondary antibody steps, which simplifies protocols, reduces assay time, and potentially decreases background noise in experimental applications .

How should researchers validate the specificity of SERPINB2 antibodies in experimental systems?

Validating antibody specificity is critical for reliable research outcomes. Based on current methodological approaches, researchers should employ the following validation strategy:

• Knockout Controls: The gold standard for validation involves comparing detection in parental cell lines versus SERPINB2 knockout lines. A specific band should be present in parental cells but absent in knockout cells at the expected molecular weight (40-47 kDa) .

• Induction Experiments: Treat cells known to express SERPINB2 (e.g., U937, K562, or HEK001) with PMA to upregulate SERPINB2 expression and compare with untreated controls. This approach confirms antibody responsiveness to changing protein levels .

• Cross-Reactivity Assessment: If the antibody claims cross-reactivity with multiple species, evaluate performance across these species. Based on immunogen sequence analysis, expected cross-reactivity may include human (100%), gorilla, monkey (92%), dog (90%), and horse (85%) .

• Loading Controls: Always include appropriate loading controls (e.g., HSP60) to ensure equal sample loading and to distinguish specific signals from loading variations .

• Multiple Detection Methods: When possible, validate findings using orthogonal detection methods (e.g., Simple Western™ alongside traditional Western blot) .

This comprehensive validation strategy ensures that observed signals genuinely represent SERPINB2 rather than non-specific binding or artifacts.

What are the optimal conditions for Western blot detection using SERPINB2 antibodies?

Successful Western blot detection of SERPINB2 requires careful attention to experimental conditions. Based on the scientific literature, the following protocol parameters have been validated:

For HRP-conjugated antibodies specifically, direct detection with appropriate substrates eliminates the need for secondary antibody incubation. When troubleshooting, researchers should systematically evaluate blocking conditions, antibody concentration, and incubation parameters to optimize signal-to-noise ratio .

How does SERPINB2 expression vary across different experimental conditions?

SERPINB2 expression shows significant variation across cell types and conditions, which has important implications for experimental design:

Cell Type Differences:

• High expression in activated monocytes/macrophages

• Detectable in conventional dendritic cells at low levels

• Absent in plasmacytoid dendritic cells, B cells, T cells, and NK cells

• Present in human epidermal keratinocyte (HEK001) and histiocytic lymphoma (U937) cell lines

Inducible Expression:

• Dramatically upregulated by PMA treatment in U937 and K562 cells

• Early response gene to epigallocatechin gallate (EGCG) treatment

• In KYSE150 cells, protein levels increased from 14-22 hours after EGCG treatment

• In KYSE510 cells, elevation occurs as early as 3 hours after treatment

Temporal Dynamics:

• Synchronized fluctuation with Caspase-3 during time course experiments, suggesting potential co-regulation with apoptotic pathways

• High induction at early time points after EGCG treatment, with reduction after 24 hours but still elevated compared to controls

These expression patterns should guide experimental design, particularly for selecting appropriate positive controls and time points for SERPINB2 detection.

What methodological approaches are recommended for immunohistochemical detection of SERPINB2?

For optimal immunohistochemical (IHC) detection of SERPINB2 in tissues, researchers should consider the following methodological approaches:

• Antibody Selection: Multiple manufacturers offer antibodies validated for IHC applications . For HRP-conjugated antibodies, direct detection systems eliminate the need for secondary antibodies, potentially reducing background in tissue sections.

• Tissue Considerations: Based on SERPINB2 expression patterns, placenta, skin, and inflammatory tissues rich in activated macrophages represent ideal positive control tissues .

• Protocol Optimization:

  • Antigen retrieval methods may be necessary, particularly for formalin-fixed tissues

  • Blocking endogenous peroxidase activity is critical when using HRP-conjugated antibodies

  • Appropriate blocking of non-specific binding sites with serum matching the host of primary antibody

• Detection Systems: For low abundance situations, signal amplification systems may be beneficial, though this is less necessary with direct HRP conjugates that already provide signal enhancement.

• Validation Strategy: Confirm specificity using parallel staining of tissues from SERPINB2 knockout models when available. Alternatively, peptide blocking experiments can help confirm specificity.

Although detailed IHC protocols specific to SERPINB2 were limited in the search results, researchers can adapt standard IHC methods using the dilution and reactivity information provided in antibody documentation .

How does SERPINB2 function in inflammation and immune regulation?

Recent research has revealed SERPINB2 as an important regulator of inflammatory processes, with implications extending beyond its canonical protease inhibitory function:

Immunoregulatory Role:
SERPINB2 knockout (SERPINB2−/−) mice exhibit dramatically enhanced Th1 immune responses compared to wild-type littermates, including approximately 6-fold higher IgG2c production and 2.5-fold more antigen-specific IFN-γ–secreting T cells following immunization . This suggests SERPINB2 functions as a natural suppressor of Th1-polarized immunity.

Cellular Mediation Mechanisms:

• SERPINB2−/− macrophages promote greater IFN-γ secretion from wild-type T cells both in vivo and in vitro

• When stimulated with anti-CD40/IFN-γ or cultured with T cells, SERPINB2−/− macrophages secrete elevated levels of Th1-promoting cytokines

• Similar enhancement of Th1-promoting cytokine secretion occurs in SERPINB2−/− myeloid antigen-presenting cells from draining lymph nodes

Disease Associations:
SERPINB2 polymorphisms or dysregulated expression have been linked to multiple inflammatory conditions, including pre-eclampsia, lupus, asthma, scleroderma, and periodontitis . These associations highlight the clinical relevance of SERPINB2's immunoregulatory functions.

Anti-Tumor Activity:
SERPINB2 has been identified as a tumor-suppressor gene involved in cell movement regulation and apoptosis, suggesting potential therapeutic applications in cancer, particularly esophageal cancer . The synchronous fluctuation with Caspase-3 suggests potential involvement in apoptotic regulation .

Understanding these multifaceted roles of SERPINB2 in inflammation and immune regulation provides important context for interpreting experimental results and developing targeted research questions.

What are the challenges and solutions for detecting low abundance SERPINB2 in complex samples?

Detection of low abundance SERPINB2 in biological samples presents several methodological challenges that can be addressed through optimized experimental approaches:

Challenges:

• Variable expression across cell types and conditions

• Background interference in complex samples

• Potentially masked epitopes due to protein interactions or modifications

• Limited sensitivity of standard detection methods

Methodological Solutions:

For particularly challenging samples, researchers might consider inducing SERPINB2 expression with PMA or other triggers where biologically appropriate, while recognizing this alters the physiological state .

How do post-translational modifications affect SERPINB2 detection and function?

Post-translational modifications (PTMs) of SERPINB2 can significantly impact both antibody recognition and protein function:

Glycosylation Effects:
SERPINB2 contains reported glycosylation sites that can affect protein conformation and epitope accessibility . Glycosylation may create steric hindrance that prevents antibody binding, particularly for antibodies raised against peptide sequences containing or adjacent to glycosylation sites.

Functional Implications:

• PTMs can regulate SERPINB2's inhibitory activity against target proteases

• Modifications may affect SERPINB2's subcellular localization between cytoplasmic and secreted pools

• PTMs potentially influence SERPINB2's interactions with binding partners

Methodological Considerations:

• Use multiple antibodies targeting different epitopes to ensure comprehensive detection

• Consider enzymatic deglycosylation treatments prior to detection to assess glycosylation effects

• When quantifying SERPINB2, be aware that PTMs can affect antibody binding affinity and thus apparent concentration measurements

• For comparative studies, ensure consistent sample preparation to maintain uniform PTM status

Research Applications:
Understanding the relationship between SERPINB2 PTMs and function offers opportunities for studying regulatory mechanisms in different physiological and pathological contexts, particularly in inflammatory conditions where SERPINB2 plays regulatory roles .

What are the key considerations for multiplex detection systems involving SERPINB2?

When incorporating SERPINB2 detection into multiplex systems, researchers should address several critical methodological considerations:

• Antibody Compatibility: Ensure that antibodies against multiple targets don't interfere with each other's binding. This is particularly important when targets might be in close proximity or part of protein complexes.

• Signal Discrimination: With HRP-conjugated SERPINB2 antibodies, carefully select substrate systems that allow discrimination from other detection channels (e.g., fluorescent, chromogenic) used in the multiplex assay.

• Cross-Reactivity Assessment: Validate that anti-SERPINB2 antibodies don't cross-react with other serpin family members that may be present in the same samples. Sequence similarity within the serpin family necessitates rigorous specificity testing .

• Sample Preparation Optimization: Different targets may require different extraction conditions. Develop extraction protocols that effectively solubilize all targets of interest while preserving their native states or epitopes.

• Quantification Calibration: For quantitative multiplex assays, ensure that the dynamic range of SERPINB2 detection is compatible with other targets being measured simultaneously.

• Timing Considerations: If temporal dynamics are important (e.g., the synchronized fluctuation with Caspase-3) , ensure all components of the multiplex system can capture relevant time points accurately.

• Validation Strategy: Validate the multiplex system against single-plex detection for each component to ensure no loss of sensitivity or specificity in the combined system.

By systematically addressing these considerations, researchers can successfully incorporate SERPINB2 detection into multiplex experimental systems for more comprehensive analysis of biological processes.

What are the emerging applications of SERPINB2 antibodies in research?

Several promising research directions are emerging for SERPINB2 antibodies:

• Immune Regulation Studies: Given SERPINB2's newly discovered role in regulating Th1 responses, antibodies against this protein are becoming valuable tools for investigating inflammatory disease mechanisms .

• Cancer Research Applications: The identification of SERPINB2 as a tumor suppressor gene opens opportunities for using these antibodies to evaluate its expression and function in various cancer types .

• Biomarker Development: SERPINB2 detection may have potential as a biomarker for certain inflammatory conditions or cancer prognoses, with HRP-conjugated antibodies offering direct detection capabilities for diagnostic development.

• Cell Signaling Investigations: The observed synchronous fluctuation with Caspase-3 suggests potential applications in studying apoptotic pathways and cell fate decisions .

• Therapeutic Target Validation: Antibodies are essential tools for validating potential therapeutic approaches targeting SERPINB2 or its regulatory pathways.

What quality control parameters should researchers evaluate when selecting SERPINB2 antibodies?

When selecting SERPINB2 antibodies for research applications, researchers should evaluate several critical quality parameters:

By thoroughly evaluating these parameters, researchers can select SERPINB2 antibodies most appropriate for their specific experimental needs, increasing the reliability and reproducibility of their research outcomes.