SPINT2 Antibody

What is SPINT2 and what are its primary biological functions?

SPINT2 (Serine Peptidase Inhibitor, Kunitz Type 2) is a 252 amino acid protein with a molecular weight of approximately 28.2 kDa that functions as a Kunitz-type serine protease inhibitor . The protein is primarily membrane-localized and exhibits inhibitory activity against several serine proteases, most notably TMPRSS2 (Transmembrane Serine Protease 2) and HGF activator .

The biological significance of SPINT2 includes:

• Regulation of epithelial cell barrier function through inhibition of matriptase

• Modulation of HGF (Hepatocyte Growth Factor) signaling through inhibition of HGF activator

• Control of TMPRSS2 activity, which has implications for viral entry mechanisms

• Tissue-specific expression patterns, with notable presence in placenta, kidney, pancreas, prostate, testis, thymus, and trachea

The protein undergoes post-translational modification, particularly glycosylation, which may affect its inhibitory function . Up to two different isoforms have been reported, suggesting tissue-specific roles for different SPINT2 variants .

How is SPINT2 expression regulated at the transcriptional level?

SPINT2 expression exhibits a tightly regulated balance with its target proteases, particularly TMPRSS2. Research has revealed common transcription factors associated with genomic loci for both SPINT2 and TMPRSS2 genes, explaining their correlated expression patterns across different cell types .

Transcriptional regulation analysis shows:

• Coregulation exists between SPINT2 and TMPRSS2, with consistent correlation across various cell types

• The highest correlation between SPINT2 and TMPRSS2 expression is observed in tissues that are targets for SARS-CoV-2 infection

• At single-cell resolution, both genes demonstrate specific co-expression in multiple cell types, corroborating the inferred coregulation

This transcriptional coregulation has significant implications for understanding how cells maintain protease/inhibitor balance in different physiological and pathological contexts.

What research methods are typically used to study SPINT2 protein interactions?

To investigate SPINT2 interactions with target proteases like TMPRSS2, researchers employ several methodological approaches:

• Co-immunoprecipitation assays: To detect physical interactions between SPINT2 and its target proteases in cell lysates

• Enzymatic inhibition assays: Measuring protease activity in the presence of recombinant SPINT2 or cellular extracts with varying SPINT2 expression

• Knockdown and overexpression studies: As demonstrated in the SARS-CoV-2 research, shRNA-mediated SPINT2 knockdown and overexpression systems help evaluate functional consequences of altered SPINT2 levels

• Pharmacological inhibition: Using specific inhibitors like Camostat mesylate to block TMPRSS2 activity can help determine if SPINT2 effects are mediated through TMPRSS2 inhibition

• Western blot analysis: To monitor expression levels of SPINT2 and potential interaction partners

In combination, these methods provide robust evidence for SPINT2's inhibitory functions and biological significance.

What criteria should researchers consider when selecting a SPINT2 antibody for their experiments?

When selecting a SPINT2 antibody for research applications, consider the following critical factors:

• Application compatibility: Verify the antibody has been validated for your specific application (Western blot, ELISA, IHC, IF)

• Species reactivity: Confirm reactivity with your experimental model species. Available SPINT2 antibodies may react with human, mouse, bovine, or other species

• Epitope recognition: Consider whether you need an antibody targeting a specific region (e.g., C-terminal, middle region) which may be important if studying specific isoforms

• Clonality: Monoclonal antibodies offer high specificity for a single epitope, while polyclonal antibodies recognize multiple epitopes, potentially providing stronger signals

• Conjugation requirements: Determine if you need unconjugated antibodies or those with specific conjugates (HRP, biotin) based on your detection system

• Validation evidence: Check for published citations demonstrating successful use in applications similar to yours

• Isoform specificity: Since SPINT2 has up to two reported isoforms, ensure the antibody can detect your isoform of interest

What validation methods should be employed to confirm SPINT2 antibody specificity?

Thorough validation of SPINT2 antibodies is essential for reliable experimental results. Implement these validation approaches:

• Positive and negative control samples: Use tissues or cell lines with known SPINT2 expression (placenta, kidney, pancreas) as positive controls, and non-expressing tissues as negative controls

• Recombinant protein controls: Test antibody against purified recombinant SPINT2 protein in Western blot or ELISA

• Knockdown/knockout validation: Compare antibody reactivity in wild-type cells versus SPINT2 knockdown cells, as demonstrated in the SARS-CoV-2 study where SPINT2 knockdown validation was performed at both transcript and protein levels

• Cross-reactivity assessment: Test against related Kunitz-type inhibitors to ensure specificity

• Peptide competition assays: Pre-incubate antibody with immunizing peptide to demonstrate signal specificity

• Multiple antibody comparison: Use antibodies targeting different epitopes of SPINT2 to confirm consistent detection patterns

• Molecular weight verification: Confirm detection at the expected molecular weight (approximately 28.2 kDa for the canonical form, with potential variation due to glycosylation)

What are the optimal protocols for SPINT2 detection in Western blot applications?

For optimal Western blot detection of SPINT2, follow these methodological considerations:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylated forms

  • Denature samples at 95°C for 5 minutes in reducing conditions

• Gel selection and transfer:

  • Use 10-12% SDS-PAGE gels for optimal separation

  • Transfer to PVDF membranes (preferred over nitrocellulose for glycosylated proteins)

  • Transfer at 100V for 60-90 minutes in cold transfer buffer containing 20% methanol

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with primary SPINT2 antibody at 1:500-1:1000 dilution overnight at 4°C

  • Wash extensively with TBST (at least 3×10 minutes)

  • Incubate with HRP-conjugated secondary antibody at 1:5000 dilution for 1 hour

• Detection considerations:

  • Due to glycosylation, SPINT2 may appear at a higher molecular weight than predicted

  • Use enhanced chemiluminescence (ECL) detection systems

  • Include positive control lysates from cells with known SPINT2 expression

• Troubleshooting guidance:

  • If detecting multiple bands, confirm specificity with knockdown studies

  • For weak signals, extend primary antibody incubation time or increase concentration

  • For high background, increase washing steps or decrease antibody concentration

How can researchers optimize immunohistochemistry protocols for SPINT2 detection in tissue samples?

Optimizing immunohistochemistry (IHC) for SPINT2 detection requires specific attention to several parameters:

• Tissue fixation and processing:

  • Use 10% neutral-buffered formalin for fixation (24-48 hours)

  • Paraffin embedding should follow standard protocols

  • Cut sections at 4-5 μm thickness for optimal staining

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) is generally effective

  • Pressure cooking for 3-5 minutes typically provides sufficient retrieval

  • Allow slides to cool in retrieval solution for 20 minutes before proceeding

• Blocking parameters:

  • Block endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes

  • Block non-specific binding with 5% normal serum from the species of secondary antibody

  • Include avidin-biotin blocking if using biotinylated detection systems

• Antibody selection and dilution:

  • Test antibodies validated specifically for IHC applications

  • Start with manufacturer's recommended dilution and optimize as needed

  • Incubate primary antibody overnight at 4°C for best results

• Detection systems:

  • Polymer-based detection systems often provide cleaner backgrounds than ABC methods

  • DAB (3,3'-diaminobenzidine) is the most common chromogen for visualization

  • Consider double staining with other markers if studying co-localization

Include positive control tissues known to express SPINT2 (placenta, kidney, pancreas) and implement appropriate negative controls (primary antibody omission, non-immune IgG substitution).

What methods should be used to quantify SPINT2 expression levels in cell and tissue samples?

Accurate quantification of SPINT2 expression requires selecting appropriate methods based on research questions:

• Western blot quantification:

  • Use densitometry software (ImageJ, Image Lab) to analyze band intensity

  • Normalize SPINT2 signal to loading controls (β-actin, GAPDH, or total protein)

  • Include concentration standards if absolute quantification is needed

  • Present results as fold-change relative to control samples

• qRT-PCR for transcript quantification:

  • Design primers spanning exon-exon junctions to avoid genomic DNA amplification

  • Normalize to validated reference genes stable in your experimental conditions

  • Calculate expression using the 2^-ΔΔCt method for relative quantification

• ELISA-based quantification:

  • Commercial ELISA kits are available for SPINT2 quantification

  • Develop standard curves using recombinant SPINT2 protein

  • Consider sandwich ELISA format using two antibodies recognizing different epitopes

• Immunohistochemistry quantification:

  • Use digital image analysis software for IHC staining quantification

  • Score based on staining intensity and percentage of positive cells

  • Consider H-score system (0-300) combining intensity and positivity percentage

  • Implement automated systems with machine learning algorithms for unbiased assessment

• Single-cell analysis:

  • Flow cytometry can quantify SPINT2 at single-cell resolution

  • Single-cell RNA-seq can reveal expression heterogeneity within populations

How can researchers study the functional relationship between SPINT2 and TMPRSS2 in experimental systems?

Investigating the SPINT2-TMPRSS2 regulatory axis requires sophisticated experimental approaches:

• Co-expression analysis:

  • Quantify SPINT2 and TMPRSS2 expression correlation across tissues and cell types

  • Analyze single-cell RNA-seq data to identify co-expressing populations

  • Implement fluorescent dual-labeling to visualize co-expression at protein level

• Genetic manipulation strategies:

  • Generate SPINT2 knockdown models using shRNA or siRNA approaches

  • Create SPINT2 overexpression systems with tagged constructs

  • Implement CRISPR/Cas9 for complete knockout studies

  • Develop inducible expression systems for temporal control of SPINT2 levels

• Protease activity measurements:

  • Use fluorogenic peptide substrates specific for TMPRSS2 to measure activity

  • Compare protease activity in SPINT2-manipulated versus control cells

  • Implement live-cell imaging with protease-activated reporters

• Binding interaction studies:

  • Perform co-immunoprecipitation to demonstrate physical interaction

  • Use surface plasmon resonance (SPR) to measure binding kinetics

  • Implement proximity ligation assays (PLA) to visualize interactions in situ

• Functional readouts:

  • In viral infection models, measure viral entry and replication as functional readouts

  • For HGF signaling, assess downstream phosphorylation events (e.g., c-Met phosphorylation)

  • Monitor epithelial barrier integrity in cell models with SPINT2 manipulation

Research has shown that SPINT2 knockdown increases TMPRSS2 gene expression compared to wild-type or scramble control cells, suggesting a feedback regulatory mechanism .

What are the common challenges and solutions when working with SPINT2 antibodies in various applications?

Researchers frequently encounter these challenges when working with SPINT2 antibodies:

How can researchers implement advanced imaging techniques to study SPINT2 localization and trafficking?

Advanced imaging approaches provide crucial insights into SPINT2 dynamics:

• Super-resolution microscopy techniques:

  • Structured Illumination Microscopy (SIM) achieves 100nm resolution for detailed membrane localization

  • Stochastic Optical Reconstruction Microscopy (STORM) provides 20nm resolution for precise colocalization studies

  • Stimulated Emission Depletion (STED) microscopy allows for live-cell super-resolution imaging

• Live-cell imaging strategies:

  • Generate SPINT2-fluorescent protein fusions (e.g., SPINT2-GFP) for real-time visualization

  • Implement photoactivatable or photoconvertible tags for pulse-chase imaging

  • Use fluorescence recovery after photobleaching (FRAP) to measure membrane dynamics

• Proximity-based interaction studies:

  • Förster Resonance Energy Transfer (FRET) to study SPINT2-TMPRSS2 interactions

  • Proximity Ligation Assay (PLA) to visualize protein-protein interactions with standard microscopy

  • Bimolecular Fluorescence Complementation (BiFC) for direct visualization of interacting proteins

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescence imaging of SPINT2 with ultrastructural context

  • Implement immunogold labeling for electron microscopy visualization

  • Use cryo-electron microscopy for near-native state visualization

• Multi-parametric imaging:

  • Co-stain with organelle markers to determine subcellular localization

  • Implement spectral unmixing for multi-protein localization studies

  • Use automated high-content imaging for quantitative localization analysis

These advanced techniques require careful validation with appropriate controls and may benefit from computational image analysis for quantitative assessment.

How is SPINT2 dysregulation implicated in viral infections, particularly SARS-CoV-2?

Research has revealed crucial roles for SPINT2 in viral infection processes:

• SARS-CoV-2 infection mechanisms:

  • SPINT2 negatively correlates with SARS-CoV-2 expression in Calu-3 and Caco-2 cell lines

  • SPINT2 knockdown significantly increases viral load in experimental systems

  • SPINT2 overexpression leads to dramatic reduction in viral load

  • The mechanism appears to involve inhibition of TMPRSS2, which is required for S protein priming

• Experimental evidence:

  • In Calu-3 cells, SPINT2 knockdown resulted in more than two-fold increase in SARS-CoV-2 positive cells

  • SPINT2 knockdown was associated with increased viral genome replication

  • Treatment with TMPRSS2 inhibitor (Camostat mesylate) abrogated the effects of SPINT2 knockdown

  • Similar results were observed in A549 cells overexpressing the SARS-CoV-2 receptor ACE2

• Clinical correlations:

  • SPINT2 was found to be down-regulated in secretory cells from COVID-19 patients

  • This suggests SPINT2 could potentially serve as a biomarker for disease susceptibility

  • The relationship may explain some COVID-19 comorbidities in patients with conditions where SPINT2 is downregulated

These findings highlight SPINT2 as a potential therapeutic target for viral infections dependent on TMPRSS2 for entry.

What is the relationship between SPINT2 expression and various pathological conditions?

SPINT2 dysregulation has been associated with multiple pathological conditions:

• Cancer associations:

  • SPINT2 is down-regulated across different types of tumors

  • Particularly evident in colon, kidney, and liver tumors

  • May function as a tumor suppressor through inhibition of proteases involved in invasion and metastasis

• Metabolic disorders:

  • Down-regulation observed in alpha pancreatic islet cells from Type 2 diabetes patients

  • This may contribute to the comorbidity observed between diabetes and COVID-19

• Gastrointestinal disorders:

  • SPINT2 gene has been associated with diarrheal diseases (DIAR3)

  • May involve dysregulation of epithelial barrier function

• Infectious disease implications:

  • SPINT2 down-regulation may increase susceptibility to certain viral infections

  • This relationship is particularly evident in SARS-CoV-2 infection models

Research into these associations suggests SPINT2 expression could serve as both a biomarker and potential therapeutic target across multiple disease contexts.

How can researchers design experiments to investigate SPINT2 as a potential therapeutic target?

Developing SPINT2-focused therapeutic strategies requires systematic experimental approaches:

• Target validation experiments:

  • Conduct dose-response studies with recombinant SPINT2 protein in disease models

  • Implement genetic rescue experiments in systems with SPINT2 deficiency

  • Use conditional knockout models to establish tissue-specific requirements

• Therapeutic modulation strategies:

  • Design small molecule compounds that enhance SPINT2 stability or function

  • Develop peptide mimetics based on SPINT2 active domains

  • Explore gene therapy approaches to restore SPINT2 expression

  • Identify compounds that upregulate endogenous SPINT2 expression

• Delivery system development:

  • Test tissue-specific delivery methods for SPINT2-based therapeutics

  • Explore nanoparticle formulations for targeted delivery

  • Investigate mRNA-based approaches for transient SPINT2 restoration

• Efficacy assessment protocols:

  • Establish clear readouts for therapeutic efficacy in disease models

  • For viral infections, measure viral entry and replication metrics

  • For cancer applications, assess changes in invasion and metastasis

  • In metabolic disorders, monitor tissue-specific functional improvements

• Safety and specificity assessment:

  • Evaluate off-target effects through protease activity profiling

  • Assess impact on related biological pathways

  • Implement toxicity screening in relevant cell and animal models

• Combination therapy exploration:

  • Test SPINT2-based approaches with existing therapies

  • For SARS-CoV-2, combine with direct-acting antivirals or TMPRSS2 inhibitors

  • In cancer, combine with conventional chemotherapy or immunotherapy

These experimental approaches should be tailored to specific disease contexts while maintaining focus on SPINT2's established biological functions.

What emerging technologies could advance our understanding of SPINT2 biology and function?

Several cutting-edge technologies hold promise for deeper insights into SPINT2 biology:

• Spatial transcriptomics and proteomics:

  • Map SPINT2 expression patterns with cellular resolution in intact tissues

  • Correlate with expression of target proteases and disease markers

  • Identify previously unknown tissue niches with significant SPINT2 activity

• Protein structure determination:

  • Apply cryo-electron microscopy to resolve SPINT2-protease complexes

  • Implement AlphaFold or RoseTTAFold for computational structure prediction

  • Use hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Single-cell multi-omics approaches:

  • Combine transcriptomics, proteomics, and epigenomics at single-cell level

  • Identify regulatory networks controlling SPINT2 expression

  • Map trajectories of SPINT2 expression changes during disease progression

• Organoid and microphysiological systems:

  • Develop tissue-specific organoids to study SPINT2 function in near-physiological contexts

  • Implement organ-on-chip technologies for dynamic functional studies

  • Create disease models incorporating patient-derived cells

• CRISPR screening approaches:

  • Perform genome-wide CRISPR screens to identify modulators of SPINT2 expression

  • Use CRISPRi/CRISPRa for targeted modulation of SPINT2 regulatory elements

  • Implement base editing for precise modification of SPINT2 sequence

These technologies will enable more comprehensive understanding of SPINT2 biology and potentially reveal new therapeutic opportunities.

How can researchers better understand the tissue-specific roles of SPINT2 in normal physiology?

Investigating tissue-specific SPINT2 functions requires specialized methodological approaches:

• Tissue-specific conditional knockout models:

  • Generate floxed SPINT2 alleles for Cre-mediated tissue-specific deletion

  • Implement inducible systems for temporal control of SPINT2 deletion

  • Analyze phenotypic consequences in multiple organ systems

• Cell type-specific transcriptomic profiling:

  • Apply single-cell RNA sequencing to tissues with significant SPINT2 expression

  • Identify cell populations with highest SPINT2 expression

  • Map co-expression networks to infer tissue-specific functions

• Organotypic culture systems:

  • Develop 3D culture models recapitulating tissue architecture

  • Manipulate SPINT2 expression in these systems

  • Assess functional outcomes relevant to specific tissues

• In vivo imaging approaches:

  • Generate SPINT2 reporter mouse models for in vivo visualization

  • Implement intravital microscopy to observe dynamic changes

  • Correlate SPINT2 expression with tissue function in real-time

• Targeted proteomics:

  • Identify tissue-specific SPINT2 interacting partners

  • Characterize post-translational modifications across tissues

  • Determine if different SPINT2 isoforms predominate in specific tissues

These approaches will help define the physiological roles of SPINT2 beyond its known pathological associations, potentially revealing new therapeutic opportunities.