SERPINI2 Antibody

What is SERPINI2 and what is its primary function in biological systems?

SERPINI2 (Serpin I2) is a member of the serpin superfamily of serine protease inhibitors. It functions primarily as an inhibitor of specific proteases, contributing to the regulation of proteolytic cascades. Serpins generally work through a mechanism involving conformational change and formation of covalent complexes with target proteases. SERPINI2 is expressed in several tissues including the pancreas and has been implicated in maintaining tissue homeostasis through protease inhibition .

What are the key characteristics that differentiate SERPINI2 from other serpin family members?

SERPINI2 is distinguished from other serpin family members like SERPINE2 (PN-1) and SERPINB2 by its:

• Tissue expression pattern: Predominantly expressed in the pancreas, unlike SERPINE2 which is expressed in multiple tissues

• Molecular structure: While sharing the conserved serpin domain structure, SERPINI2 has unique regions that determine its specific protease targets

• Function: SERPINI2 has distinct protease inhibition profiles compared to other serpins like SERPINE2, which inhibits thrombin, trypsin, and urokinase

• Chromosomal location: Located on a different chromosome than other serpin family members

• Evolutionary conservation: Shows different patterns of conservation across species compared to other serpins

How should researchers store and handle SERPINI2 antibodies to maintain optimal activity?

For optimal antibody performance, researchers should follow these evidence-based storage and handling protocols:

• Temperature conditions: Store at -20 to -70°C for long-term storage (up to 12 months from receipt date)

• Short-term storage: 2 to 8°C under sterile conditions after reconstitution (up to 1 month)

• Medium-term storage: -20 to -70°C under sterile conditions after reconstitution (up to 6 months)

• Avoid repeated freeze-thaw cycles: Use a manual defrost freezer to prevent antibody degradation

• Aliquoting: Divide reconstituted antibody into single-use aliquots to minimize freeze-thaw cycles

• Sterile handling: Maintain sterile conditions when handling the reconstituted antibody

This handling protocol is based on established guidelines for serpin antibodies and ensures retention of antibody specificity and sensitivity .

What are the validated applications for SERPINI2 antibodies in experimental research?

Based on research with serpin family antibodies, the following applications have been validated for SERPINI2 antibody:

Researchers should perform optimization experiments to determine the ideal concentration for their specific experimental conditions .

How can researchers optimize Western blot protocols specifically for SERPINI2 detection?

For optimal SERPINI2 detection by Western blot, follow this methodological approach:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for cell/tissue lysis

  • Heat samples at 95°C for 5 minutes in reducing sample buffer

• Gel electrophoresis:

  • Use 10% SDS-PAGE for optimal resolution (SERPINI2 predicted size: ~45 kDa)

  • Load 30-50 μg of total protein per lane as demonstrated in successful detection of similar serpins

• Transfer and blocking:

  • Transfer to PVDF membrane at 100V for 60-90 minutes

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

• Primary antibody incubation:

  • Dilute SERPINI2 antibody 1:1000-1:2000 in blocking solution

  • Incubate overnight at 4°C with gentle rocking

• Detection optimization:

  • Use HRP-conjugated secondary antibody (1:5000)

  • Employ enhanced chemiluminescence detection

  • For weak signals, consider signal enhancement systems or longer exposure times

• Controls:

  • Include positive control tissue known to express SERPINI2

  • Use β-actin or GAPDH as loading controls

  • Include a negative control lacking primary antibody

This protocol is based on successful detection of similar serpin family proteins as demonstrated in the literature .

What are the recommended protocols for immunohistochemistry using SERPINI2 antibodies?

For optimal immunohistochemical detection of SERPINI2:

• Tissue preparation:

  • Fix tissues in 10% neutral-buffered formalin

  • Embed in paraffin and section at 4-6 μm thickness

• Antigen retrieval (critical step):

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

  • Allow slides to cool slowly to room temperature (approximately 20 minutes)

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

  • Block non-specific binding with 5% normal serum for 30 minutes

  • Apply SERPINI2 primary antibody (1:100-1:200 dilution) and incubate overnight at 4°C

• Detection system:

  • Use biotin-streptavidin-HRP or polymer-based detection systems

  • Develop with DAB substrate for 2-5 minutes (monitor under microscope)

  • Counterstain with hematoxylin for 30 seconds

• Controls and validation:

  • Include pancreatic tissue as positive control

  • Perform antibody validation using SERPINI2 knockdown tissues

  • Include isotype control to assess non-specific binding

This protocol is adapted from successful IHC methods used for other serpin family members and should be optimized for specific tissue types .

How can researchers verify the specificity of SERPINI2 antibodies in their experimental systems?

To validate SERPINI2 antibody specificity, implement these methodological approaches:

• Cross-reactivity assessment:

  • Test antibody against recombinant SERPINI2 and related serpins (SERPINA1, SERPINE1, SERPINB2)

  • Perform peptide competition assays using the immunizing peptide

  • Compare staining patterns with multiple SERPINI2 antibodies raised against different epitopes

• Genetic validation:

  • Use SERPINI2 knockout or knockdown models (siRNA, shRNA, CRISPR) as negative controls

  • Perform recovery experiments with SERPINI2 overexpression in knockout models

• Western blot validation:

  • Confirm single band at expected molecular weight (~45 kDa)

  • Test multiple tissue types with known differential SERPINI2 expression

  • Compare with mRNA expression data from public databases

• Immunoprecipitation validation:

  • Perform IP followed by mass spectrometry to confirm target identity

  • Conduct reverse IP using purified SERPINI2 protein

Similar validation approaches have been successfully applied to other serpin antibodies, as seen with the validation of SERPINE2 antibodies that showed no cross-reactivity with human Serpin A1, A3, A4, A5, or mouse Serpin C1, D1, F2 .

What potential artifacts or false positives should researchers be aware of when using SERPINI2 antibodies?

Researchers should be vigilant about these potential artifacts and implement appropriate controls:

• Cross-reactivity issues:

  • Other serpin family members (particularly SERPINI1) due to sequence homology

  • Non-specific binding to denatured proteins in fixed tissues

  • Cross-reactivity with species-specific variants

• Technical artifacts:

  • Edge effects in immunohistochemistry

  • Incomplete antigen retrieval leading to false negatives

  • Overfixation masking epitopes

  • Background from endogenous biotin in biotin-based detection systems

• Sample-specific considerations:

  • Endogenous peroxidase activity in tissues rich in blood cells

  • Autofluorescence in specific tissues (particularly formaldehyde-fixed tissues)

  • Non-specific binding to necrotic tissue areas

• Recommended controls:

  • Pre-absorption with immunizing peptide

  • SERPINI2 knockout/knockdown samples

  • Isotype control antibodies at the same concentration

  • Secondary antibody-only controls

These considerations are based on documented challenges with serpin antibodies and general immunodetection principles .

How can SERPINI2 antibodies be utilized in multiplex immunofluorescence for co-localization studies?

For successful multiplex immunofluorescence with SERPINI2 antibodies, follow this methodological approach:

• Antibody panel design:

  • Select antibody pairs raised in different host species (e.g., rabbit anti-SERPINI2 with mouse anti-cell marker)

  • Alternatively, use directly conjugated primary antibodies with different fluorophores

  • Validate each antibody individually before multiplexing

• Sequential staining protocol:

  • Apply SERPINI2 antibody first (1:100 dilution)

  • Incubate overnight at 4°C

  • Apply fluorophore-conjugated secondary antibody

  • Block with excess unconjugated host IgG before applying next primary antibody

  • Repeat for additional markers

• Spectral considerations:

  • Choose fluorophores with minimal spectral overlap

  • Include single-color controls for spectral unmixing

  • Consider tyramide signal amplification for weak signals

• Image acquisition and analysis:

  • Use confocal microscopy with sequential scanning

  • Perform colocalization analysis using Pearson's or Mander's coefficients

  • Conduct quantitative analysis using automated image analysis software

This approach enables simultaneous visualization of SERPINI2 with cellular markers or other serpins to study spatial relationships and potential functional interactions .

What strategies can researchers employ to investigate SERPINI2 protein-protein interactions using antibody-based approaches?

To study SERPINI2 protein interactions, implement these advanced methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Lyse cells under native conditions (non-denaturing buffer)

  • Pre-clear lysate with protein A/G beads

  • Immunoprecipitate with anti-SERPINI2 antibody (5-10 μg per mg of protein)

  • Analyze precipitated complexes by Western blot using antibodies against suspected binding partners

  • Include IgG control and reverse Co-IP for validation

• Proximity ligation assay (PLA):

  • Fix cells/tissues with 4% paraformaldehyde

  • Apply primary antibodies against SERPINI2 and potential interaction partner

  • Use species-specific PLA probes

  • Perform rolling circle amplification and detection

  • Quantify interaction signals as fluorescent spots per cell

• Chromatin immunoprecipitation (ChIP) for transcription factor studies:

  • Cross-link protein-DNA complexes with formaldehyde

  • Sonicate to fragment chromatin

  • Immunoprecipitate with anti-SERPINI2 antibody

  • Reverse cross-links and analyze bound DNA by qPCR or sequencing

• FRET/FLIM analysis:

  • Tag SERPINI2 and potential partners with appropriate fluorophores

  • Measure energy transfer using fluorescence lifetime imaging

  • Calculate FRET efficiency to determine molecular proximity

These approaches have been successfully used to study protein interactions of other serpin family members and can be adapted for SERPINI2 research .

How can researchers apply SERPINI2 antibodies in studying disease models and pathological processes?

For investigating SERPINI2 in disease models, implement these methodological approaches:

• Disease model characterization:

  • Quantify SERPINI2 expression changes using immunoblotting and immunohistochemistry

  • Compare SERPINI2 levels between healthy and diseased tissues using standardized scoring systems

  • Correlate SERPINI2 expression with disease progression markers

• Therapeutic intervention assessment:

  • Monitor SERPINI2 levels before and after treatment

  • Use tissue microarrays for high-throughput analysis across multiple patients/samples

  • Correlate changes with clinical outcomes

• Mechanistic studies in disease models:

  • Perform SERPINI2 knockdown/overexpression in disease-relevant cell lines

  • Use SERPINI2 antibodies to neutralize function in ex vivo tissue models

  • Study SERPINI2-dependent signaling pathways using phospho-specific antibodies

• Experimental design considerations:

  • Include time-course analyses to capture dynamic changes

  • Use multiple antibody-based techniques for validation

  • Consider tissue-specific expression patterns when designing experiments

This approach is based on successful studies of other serpins in disease contexts, such as SERPINE2's role in airway remodeling in asthma, where antibody treatment attenuated airway wall thickening and reduced MMP-9 and TIMP-1 expression .

What are common issues researchers encounter with SERPINI2 antibodies and how can they be resolved?

Here are methodological solutions for common problems with SERPINI2 antibody applications:

These solutions are based on general antibody troubleshooting approaches and specific experiences with serpin family antibodies .

How can researchers determine the optimal antibody concentration for different experimental applications?

For rigorous optimization of SERPINI2 antibody concentration:

• Western blot titration:

  • Prepare a dilution series (1:500, 1:1000, 1:2000, 1:5000, 1:10000)

  • Use consistent protein amount and exposure time

  • Select concentration providing specific signal with minimal background

  • Typical optimal range for SERPINI2: 1:1000-1:2000 as observed with similar serpins

• Immunohistochemistry/Immunofluorescence optimization:

  • Create an antibody dilution matrix (1:50, 1:100, 1:200, 1:500, 1:1000)

  • Test each dilution on positive control tissue

  • Assess signal-to-noise ratio quantitatively if possible

  • Consider titrating antigen retrieval conditions simultaneously

• Quantitative analysis approach:

  • Plot signal-to-noise ratio against antibody concentration

  • Calculate optimal concentration using non-linear regression

  • Determine the minimum antibody concentration that provides 80-90% of maximum specific signal

• Experimental validation:

  • Confirm optimal concentration across multiple tissue types

  • Verify reproducibility between different antibody lots

  • Document optimization parameters for laboratory protocols

This systematic approach ensures reliable and reproducible results while minimizing reagent usage and non-specific binding .

How can SERPINI2 antibodies be utilized in single-cell analysis techniques?

For applying SERPINI2 antibodies in cutting-edge single-cell techniques:

• Mass cytometry (CyTOF) applications:

  • Conjugate SERPINI2 antibody with rare earth metals

  • Optimize staining concentration (typically 1:100 starting dilution)

  • Include in panels with other serpin family members and cellular markers

  • Analyze data using dimensionality reduction techniques (tSNE, UMAP)

• Single-cell Western blot:

  • Adapt conventional Western protocols for microfluidic platforms

  • Reduce antibody concentration by 50% compared to standard Western blot

  • Extend incubation times to ensure diffusion in microchannels

  • Quantify signal at single-cell level using specialized imaging systems

• Imaging mass cytometry:

  • Use metal-tagged SERPINI2 antibodies on tissue sections

  • Optimize antibody concentration through titration experiments

  • Ablate tissue with laser and analyze metal tags by mass spectrometry

  • Create high-dimensional tissue maps of SERPINI2 expression

• Multiplex ion beam imaging:

  • Label SERPINI2 antibody with isotopically pure elemental metals

  • Apply to tissue sections with other markers of interest

  • Image using secondary ion mass spectrometry

  • Achieve subcellular resolution of SERPINI2 localization

These emerging techniques allow researchers to study SERPINI2 expression and localization with unprecedented single-cell resolution and in the context of multiple other markers simultaneously.

What considerations are important when selecting SERPINI2 antibodies for therapeutic development and translational research?

For translational applications of SERPINI2 antibodies, researchers should consider:

• Antibody characteristics for therapeutic development:

  • Species cross-reactivity (particularly mouse/human for preclinical models)

  • Epitope location relative to functional domains

  • Neutralizing vs. non-neutralizing activity

  • Antibody isotype and effector functions

  • Humanization potential for clinical applications

• Validation requirements for translational research:

  • Comprehensive specificity testing against related serpins

  • Functional validation (inhibition of enzymatic activity)

  • Dose-response characterization

  • Tissue penetration assessment

  • Pharmacokinetic/pharmacodynamic profiling

• Production and scale-up considerations:

  • Clone stability and expression levels

  • Purification efficiency and yield

  • Formulation stability

  • Binding kinetics (kon/koff rates)

  • Thermal stability and aggregation propensity

• Regulatory and clinical development path:

  • GMP production requirements

  • Toxicology study design

  • Biomarker development for patient stratification

  • Companion diagnostic potential

This approach is informed by successful therapeutic antibody development against other serpin targets, such as the monoclonal antibody against SERPINE2 that showed potential as a therapeutic agent in asthmatic airway remodeling .