serpine3 Antibody

What is SERPINE3 and why is it important in research?

SERPINE3 (Serpin Family E Member 3) is a serine protease inhibitor belonging to the serpin superfamily. In humans, the canonical protein consists of 424 amino acid residues with a molecular mass of approximately 47 kDa . SERPINE3 functions as a secreted protein and is suspected to inhibit serine proteases through the characteristic serpin mechanism of forming stable complexes with target proteases, thereby preventing their interaction with substrates .

Research importance stems from SERPINE3's specific expression patterns and evolutionary conservation across species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken . Recent studies have particularly highlighted its essential role in vision-related functions, as evidenced by its independent loss in 18 lineages that do not primarily rely on vision for survival .

What are the primary applications for SERPINE3 antibodies in laboratory research?

SERPINE3 antibodies serve multiple critical functions in research laboratories focused on protein detection and characterization. The primary applications include:

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of SERPINE3 in biological samples

• Western Blot (WB): For determining protein expression levels and molecular weight confirmation

• Immunohistochemistry (IHC): For localizing SERPINE3 in tissue sections, particularly in paraffin-embedded samples

Additionally, immunofluorescence techniques have been employed to study SERPINE3's cellular and subcellular localization, especially in retinal tissues. In situ hybridization (ISH) and fluorescent in situ hybridization (FISH) are complementary techniques used to detect SERPINE3 mRNA expression patterns .

How is SERPINE3 protein structure characterized, and what are its key functional domains?

SERPINE3 follows the characteristic serpin fold structure common to the serpin superfamily. Three-dimensional structure prediction using AlphaFold2 has provided insights into SERPINE3's conformation, allowing comparison with homologous chains of existing crystal structures of related serpins .

Key functional domains include:

• Signal peptide: Required for secretion (predicted using SignalP 5.0)

• Reactive site loop (RSL): Critical for protease inhibition and specificity

• β-sheet structure: Essential for the conformational change during protease inhibition

Post-translational modifications, particularly glycosylation, have been identified in SERPINE3, potentially affecting its function and stability . Up to two different isoforms have been reported for human SERPINE3, indicating potential functional diversity .

What are the common synonyms and related proteins to SERPINE3?

When searching literature databases or ordering antibodies, researchers should be aware of various nomenclature used for SERPINE3:

• Serpin peptidase inhibitor, clade E member 3

• Nexin-related serine protease inhibitor

• Serpin E3

• Plasminogen activator inhibitor type 1-related protein

Related proteins in the serpin superfamily include:

• SERPINE1 (PAI-1): Plasminogen activator inhibitor type 1

• SERPINE2 (PN-1): Protease nexin-1

• SerpinB3: While in a different clade, shares functional similarities as a serine protease inhibitor

Understanding these relationships is crucial for interpreting research findings and selecting appropriate experimental controls.

How is SERPINE3 expression regulated in different tissues, and what are its expression patterns in the retina?

SERPINE3 exhibits tissue-specific expression patterns with particularly notable expression in the retinal pigment epithelium (RPE). According to eyeIntegration and plae database analyses, SERPINE3 shows distinct expression patterns in human and macaque retinas .

In zebrafish, in situ hybridization studies using probes spanning coding exons 3-8 have revealed serpine3 expression patterns in the eye, particularly after lens removal . The specific expression in RPE cells suggests a specialized function in maintaining retinal homeostasis and visual function.

Regulatory mechanisms controlling SERPINE3 expression remain incompletely understood, though evolutionary analysis suggests selection pressure may be relaxed in species that do not primarily rely on vision, as evidenced by RELAX analysis from the HyPhy suite (selection intensity parameter K<1) .

What methodological considerations should be addressed when designing experiments to study SERPINE3 function?

When investigating SERPINE3 function, researchers must consider several critical methodological aspects:

• Genetic Models: CRISPR-mediated homologous recombination has been successfully employed to generate Serpine3-VenusGFP knockin mice, replacing exon 2 with a Venus reporter. This approach enables tracking of expression while disrupting protein function .

• Functional Assays: Protease activity assays using fluorescently-labeled peptide substrates allow screening of SERPINE3 against major classes of proteases to identify specific targets .

• Structural Analysis: For structure-function studies, researchers should consider both sequence analysis (using tools like DNASTAR Lasergene) and structural prediction (through AlphaFold2) to identify key functional regions and potential epitopes .

• Tissue Analysis: When analyzing retinal phenotypes in model organisms, combinations of histological approaches (H&E staining), immunohistochemistry for tight junction markers (ZO-1), and photoreceptor-specific markers provide comprehensive assessment of structural integrity .

What challenges exist in developing specific antibodies against SERPINE3, and how can they be overcome?

Developing highly specific antibodies against SERPINE3 presents several challenges:

• Cross-reactivity with related serpins: The serpin superfamily contains structurally similar members that may share epitopes, potentially resulting in antibody cross-reactivity.

• Distinguishing isoforms: With up to two reported isoforms of SERPINE3, targeting isoform-specific regions is essential for discriminating between variants .

• Species specificity: While SERPINE3 is conserved across species, sequence variations may affect antibody recognition across different experimental models.

These challenges can be overcome through:

• Epitope mapping: Identifying unique, exposed epitopes using software such as DNASTAR Lasergene and comparing with three-dimensional structures to select distinctive regions .

• Peptide-based immunization: Using synthetic peptides corresponding to unique SERPINE3 regions for immunization, similar to approaches used for SerpinB3 antibody generation .

• Extensive validation: Confirming antibody specificity through multiple techniques including ELISA against purified proteins, Western blotting, and immunohistochemistry with appropriate positive and negative controls .

What is the evolutionary significance of SERPINE3 loss in vision-independent lineages?

The independent loss of SERPINE3 in 18 lineages that typically do not primarily rely on vision represents a striking example of convergent gene loss correlated with ecological adaptation . This pattern strongly predicts a vision-related function for SERPINE3.

Evolutionary analyses using RELAX from the HyPhy suite have been employed to test whether selection pressure is relaxed (K<1) or intensified (K>1) in species with unclear SERPINE3 loss status or in clades where many close relatives have lost the gene . These analyses provide insights into the timing and selective pressures driving SERPINE3 conservation or loss.

The convergent loss pattern also offers a powerful example of how comparative genomics can reveal functional insights about genes with previously unknown functions. This evolutionarily-informed approach to gene function prediction represents an innovative strategy that complements traditional experimental methods .

What are the recommended protocols for using SERPINE3 antibodies in immunohistochemistry?

For optimal immunohistochemical detection of SERPINE3, researchers should follow these methodological guidelines:

• Tissue Preparation:

  • Fix tissues in 4% paraformaldehyde in 0.1M PBS

  • For eye tissue, remove the lens before embedding

  • Embed in gelatin/sucrose for cryosectioning or process for paraffin embedding

  • Section at 14μm thickness for optimal antibody penetration

• Antibody Dilution and Incubation:

  • Use anti-SERPINE3 antibodies at 1:200 dilution (may require optimization)

  • Incubate overnight at 4°C in a humidified chamber

  • For commercial antibodies like ab188155, validated protocols exist for human colon tissue

• Detection Systems:

  • For chromogenic detection, use appropriate secondary antibodies conjugated to enzymes like HRP or AP

  • For fluorescent detection, use fluorophore-conjugated secondary antibodies or amplification systems like TSA Plus Cy3/Cy5 kit

• Controls:

  • Include positive controls (tissues known to express SERPINE3)

  • Include negative controls (omission of primary antibody)

  • Consider using knockout/knockdown tissues as specificity controls

How should SERPINE3 antibody specificity be validated for research applications?

Thorough validation of SERPINE3 antibodies is essential for generating reliable research data. A comprehensive validation strategy includes:

• ELISA-based validation:

  • Coat plates with recombinant SERPINE3 protein (10μg/mL in carbonate buffer pH 9.6)

  • Block with 5% skimmed milk in PBS

  • Test antibody at multiple concentrations (typically 0.5-5μg/mL)

  • Include related proteins (e.g., other serpins) to assess cross-reactivity

• Western Blot validation:

  • Run purified SERPINE3 (4ng) on 12% polyacrylamide gels

  • Transfer to nitrocellulose membrane (1h 30min at 390mA)

  • Block with 5% ECL blocking agent in 0.1% PBST

  • Incubate with anti-SERPINE3 antibody (5μg/mL) overnight at 4°C

  • Verify band size matches expected 47kDa

• Immunocytochemistry/Immunohistochemistry validation:

  • Test on cell lines with known SERPINE3 expression

  • Compare with mRNA expression patterns from in situ hybridization

  • Assess subcellular localization consistency with predicted cellular location from DeepLoc 1

• Knockout/knockdown controls:

  • When possible, use SERPINE3 knockout/knockdown samples as negative controls

  • For mouse studies, Serpine3Gfp/Gfp models provide excellent validation resources

What are the optimal conditions for Western blot detection of SERPINE3?

For optimal Western blot detection of SERPINE3, researchers should follow this protocol:

• Sample Preparation:

  • For recombinant protein: 4ng of purified protein is typically sufficient

  • For tissue/cell lysates: Extract in RIPA buffer with protease inhibitors

  • Denature samples in reducing buffer (containing β-mercaptoethanol)

• Gel Electrophoresis:

  • Use 12% polyacrylamide gels for optimal resolution

  • Run at 100-120V until the dye front reaches the bottom

• Transfer Conditions:

  • Transfer to nitrocellulose membrane

  • Use 390mA for 1h 30min for efficient transfer of the 47kDa protein

• Blocking and Antibody Incubation:

  • Block with 5% ECL blocking agent in 0.1% PBST

  • Use primary antibody at 5μg/mL concentration

  • Incubate overnight at 4°C for optimal signal-to-noise ratio

• Detection:

  • Use HRP-conjugated secondary antibodies

  • Develop using enhanced chemiluminescence (ECL) reagents

  • Expected band size: 47kDa (canonical form)

• Troubleshooting:

  • If background is high, increase blocking time or detergent concentration

  • If signal is weak, extend primary antibody incubation or increase concentration

  • If multiple bands appear, verify with knockout controls or peptide competition

What techniques are recommended for investigating SERPINE3 function in retinal tissues?

For comprehensive analysis of SERPINE3 function in retinal tissues, researchers should consider these specialized techniques:

• In Situ Hybridization:

  • Design probes spanning multiple coding exons (e.g., exons 3-8)

  • Add restriction enzyme cut sites to primers for cloning

  • Clone into appropriate vectors (e.g., pCRII-topo)

  • Transcribe antisense probes with specific polymerases (e.g., SP6)

  • Use DIG-labeled NTP mix for detection

  • Perform hybridization and washing steps at 60°C

• Fluorescent In Situ Hybridization (FISH):

  • After quenching, wash sections in PBS

  • Block with 2% blocking reagent in MABT

  • Incubate with anti-digoxigenin-POD (1:500)

  • Detect signal with TSA Plus Cy3/Cy5 kit

• Retinal Phenotype Analysis:

  • Perform hematoxylin and eosin staining for general retinal architecture

  • Use anti-ZO-1 staining to assess tight junction integrity

  • Employ photoreceptor-specific markers for cell-type specific analysis

• Functional Protease Assays:

  • Use fluorescently-labeled peptide substrates

  • Screen against all major classes of proteases

  • Identify specific protease targets of SERPINE3

• Genetic Models:

  • CRISPR-mediated knockin of reporter genes (e.g., Venus)

  • Replace critical exons (e.g., exon 2) to disrupt function

  • Analyze resulting phenotypes to infer function

How can SERPINE3 antibodies be utilized in vision research and ophthalmology studies?

SERPINE3 antibodies provide valuable tools for vision research and ophthalmological studies in several key applications:

• RPE Cell Marker: As SERPINE3 shows specific expression in retinal pigment epithelium (RPE), antibodies against this protein serve as valuable markers for identifying and studying this critical cell population in both normal physiology and disease states .

• Retinal Degeneration Models: SERPINE3 antibodies enable tracking of protein expression changes during retinal degeneration in various models. The Serpine3Gfp/Gfp mouse model has demonstrated that loss of SERPINE3 function leads to retinal degeneration, suggesting that antibody-based monitoring of SERPINE3 levels could serve as a biomarker for retinal health .

• Therapeutic Target Validation: As retinal function appears dependent on proper SERPINE3 activity, antibodies that can modulate its function (activating or inhibiting) might serve as potential therapeutic agents or proof-of-concept tools for vision disorders.

• Comparative Vision Studies: Given the evolutionary pattern of SERPINE3 loss in non-vision-dependent species, antibodies against this protein can facilitate comparative studies across species with varying visual capabilities .

What role might SERPINE3 play in retinal diseases, and how can antibodies help investigate this?

The discovery that SERPINE3 is an RPE-specific protease inhibitor critical for retinal function suggests potential involvement in various retinal pathologies:

• Age-related Macular Degeneration (AMD): As AMD involves dysfunction of the RPE, SERPINE3 antibodies can help investigate whether altered protease inhibition contributes to disease progression.

• Inherited Retinal Degenerations: The retinal degeneration observed in Serpine3Gfp/Gfp mice suggests SERPINE3 dysfunction might contribute to certain inherited retinal diseases. Antibodies enable screening of patient samples for altered expression or localization .

• Protease-Mediated Retinal Damage: SERPINE3 likely regulates specific proteases in the retinal microenvironment. Antibodies can help identify these target proteases through co-immunoprecipitation studies and investigate their dysregulation in disease states .

• Diagnostic Applications: Developing sensitive ELISA assays using SERPINE3 antibodies might provide diagnostic tools for detecting retinal damage through measurement of released SERPINE3 in accessible fluids like aqueous humor.

What techniques combine SERPINE3 antibodies with other methodologies for comprehensive functional analysis?

Advanced research on SERPINE3 often requires integrating antibody-based approaches with complementary methodologies:

• Antibody-Based Proteomics with Transcriptomics:

  • Combine immunohistochemistry/immunofluorescence with RNA-seq or in situ hybridization

  • Correlate protein localization with gene expression patterns

  • Identify potential regulatory mechanisms governing SERPINE3 expression

• Structure-Function Studies:

  • Use epitope-specific antibodies targeting different regions (similar to SerpinB3 approaches)

  • Assess functional consequences of blocking specific domains

  • Correlate with structural predictions from AlphaFold2 and homology models

• Genetic Model Systems with Antibody Validation:

  • CRISPR-engineered reporter systems (e.g., Serpine3-VenusGFP)

  • Validate reporter expression patterns with antibody staining

  • Assess functional consequences of gene modification

• Evolutionary Analysis with Comparative Immunohistochemistry:

  • Apply SERPINE3 antibodies across species with varying visual dependencies

  • Correlate expression patterns with evolutionary analysis (RELAX from HyPhy suite)

  • Identify convergent adaptation patterns