SPINK9 Antibody

What is SPINK9 and what is its molecular function in human tissue?

SPINK9 (Serine Peptidase Inhibitor, Kazal Type 9) is a keratinocyte-derived cationic peptide predominantly expressed in the upper layers of palmar-plantar epidermis. It functions as a serine protease inhibitor with specific inhibitory activity against kallikrein-related peptidase 5 (KLK5). The protein is constitutively secreted by SPINK9-expressing cells and plays important roles in maintaining the physical and immunological barrier functions of the skin. SPINK9 has a molecular weight of approximately 9 kDa and consists of 86 amino acids .

What tissues should I examine when studying SPINK9 expression patterns?

When investigating SPINK9 expression patterns, prioritize skin tissue samples, particularly from palmar-plantar regions where SPINK9 is most abundantly expressed. Immunohistochemistry (IHC) has successfully detected SPINK9 in human skin tissue, human liver tissue, and human liver cancer tissue . For comprehensive expression analysis, consider examining:

• Epidermal layers with attention to differentiation stages

• Palmar-plantar epidermis versus other skin sites

• Normal versus diseased skin tissues

• Epithelial tissues from different organs

For optimal results, use antigen retrieval with TE buffer pH 9.0 or alternatively citrate buffer pH 6.0 when performing IHC on formalin-fixed, paraffin-embedded tissues .

What are the optimal storage conditions for maintaining SPINK9 antibody activity?

For maximum stability and preservation of SPINK9 antibody activity, implement the following storage protocol:

After thawing aliquots for use, keep on ice during experiments and avoid repeated freeze-thaw cycles that can degrade antibody performance. Some SPINK9 antibody products contain 0.1% BSA in smaller (20μl) sizes, which enhances stability .

What dilution ranges are recommended for SPINK9 antibodies in common applications?

The optimal dilution of SPINK9 antibodies varies by application and specific antibody product. Use the following guidelines as starting points, but always optimize for your specific experimental conditions:

When using these antibodies for the first time in any application, perform a dilution series to determine optimal concentration for your specific tissue samples or cell types. The antibody concentration needed may vary depending on antigen abundance and accessibility in different sample types .

How can I validate the specificity of SPINK9 antibodies for my experimental system?

To rigorously validate SPINK9 antibody specificity in your experimental system, implement a multi-step validation protocol:

• Positive and negative tissue controls: Use known SPINK9-expressing tissues (palmar-plantar epidermis) as positive controls and tissues with minimal expression as negative controls.

• Knockdown/knockout validation: If possible, test the antibody on samples where SPINK9 has been knocked down (siRNA) or knocked out (CRISPR/Cas9) to confirm signal reduction.

• Antigen pre-absorption test: Pre-incubate the antibody with excess recombinant SPINK9 protein before application to samples; this should significantly reduce specific staining.

• Multiple antibody comparison: Use at least two different SPINK9 antibodies targeting different epitopes to confirm consistent patterns.

• Western blot correlation: Confirm that IHC or IF patterns correlate with protein molecular weight and expression levels in Western blots from the same samples.

Document each validation step systematically with appropriate controls to establish confidence in antibody specificity before proceeding with experimental analyses .

What experimental approaches can effectively study SPINK9's role in EGFR transactivation?

To investigate SPINK9's role in EGFR transactivation, implement these methodological approaches:

• Stimulation assays: Treat human keratinocytes with recombinant SPINK9 (rSPINK9) at varying concentrations (1-100 nM) and time points (5-60 minutes) to establish dose-response and temporal activation patterns of EGFR.

• Phosphorylation detection: Monitor EGFR phosphorylation status using phospho-specific antibodies via Western blotting, ELISA, or phospho-flow cytometry after SPINK9 treatment.

• Inhibitor studies: Use metalloproteinase inhibitors (e.g., GM6001) and EGFR-blocking antibodies to determine the involvement of ADAMs in SPINK9-mediated EGFR transactivation.

• Purinergic receptor antagonism: Apply classical purinergic receptor antagonists (oxidized ATP and pyridoxalphosphate-6-azophenyl-2',4',-disulfonic acid) to examine their role in SPINK9-induced EGFR transactivation.

• Downstream signaling analysis: Assess activation of ERK1/2, AKT, and other EGFR downstream effectors to confirm functional pathway activation.

• Cell migration assays: Quantify keratinocyte migration using scratch assays or transwell migration systems following SPINK9 stimulation with and without EGFR or metalloprotease inhibitors.

• siRNA knockdown: Target specific ADAMs or purinergic receptors with siRNA to identify which specific family members mediate SPINK9's effects .

How can I distinguish between SPINK9's protease inhibitory function and its EGFR signaling role?

Distinguishing between SPINK9's dual functions requires a comprehensive methodological approach:

• Domain-specific mutants: Generate SPINK9 mutants with alterations in:

  • The protease inhibitory domain (abolishing KLK5 inhibition)

  • Regions potentially involved in EGFR pathway activation

  Test these mutants in parallel functional assays to separate the two activities.

• Functional assays for protease inhibition:

  • KLK5 enzymatic activity assays using fluorogenic substrates

  • In vitro protease inhibition assays with purified components

  • Zymography to visualize inhibition of proteolytic activity

• EGFR signaling-specific assays:

  • EGFR phosphorylation studies

  • ADAM activation assays

  • Purinergic receptor involvement using specific antagonists

• Temporal separation studies: Determine if these functions occur with different kinetics or under different conditions by time-course experiments.

• Competitive inhibition: Use excess KLK5 to sequester SPINK9's protease inhibitory function and determine if EGFR activation remains intact.

• Co-localization studies: Perform immunofluorescence to determine if SPINK9 co-localizes with KLK5 versus EGFR/ADAMs in different cellular compartments .

What are the recommended methodologies for evaluating SPINK9 antibody performance across different experimental platforms?

To comprehensively evaluate SPINK9 antibody performance across platforms:

• Epitope mapping correlation:

  • Determine the exact epitope recognized by each antibody (if known)

  • Antibodies targeting AA 20-86 versus the middle region may perform differently

  • Correlate recognition site with accessibility in different experimental conditions

• Cross-platform validation matrix:

• Sample preparation impact:

  • Compare fresh frozen versus FFPE tissues

  • Test different fixatives (formalin, methanol, acetone)

  • Evaluate various antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

• Batch-to-batch consistency testing:

  • Maintain reference samples for quality control

  • Document lot-specific performance metrics

  • Calculate coefficient of variation between batches

How should I troubleshoot weak or absent SPINK9 signal in immunohistochemistry applications?

When encountering weak or absent SPINK9 signal in IHC, implement this systematic troubleshooting approach:

• Antigen retrieval optimization:

  • Compare heat-induced epitope retrieval (HIER) methods:

    • TE buffer pH 9.0 (primary recommendation)

    • Citrate buffer pH 6.0 (alternative method)

  • Test different retrieval durations (10-30 minutes)

  • Optimize temperature (95-125°C)

• Antibody concentration adjustment:

  • For weak signals, try more concentrated antibody (1:20-1:50)

  • Create a dilution series (1:20, 1:50, 1:100, 1:200, 1:500)

  • Extend primary antibody incubation time (overnight at 4°C)

• Detection system enhancement:

  • Switch to a more sensitive detection system (e.g., polymer-based)

  • Use amplification systems like tyramide signal amplification

  • Consider fluorescent detection for low abundance targets

• Tissue quality assessment:

  • Verify tissue fixation quality with control antibodies

  • Check tissue block age (older blocks may require more aggressive retrieval)

  • Use freshly cut sections (avoid stored slides)

• Positive control verification:

  • Always include known positive tissue (skin samples)

  • Process control tissues alongside test samples

What controls are critical when studying SPINK9's involvement in cell migration processes?

When investigating SPINK9's role in cell migration, implement these essential controls:

• Stimulation controls:

  • Vehicle-only treatment control

  • Heat-inactivated SPINK9 (denatured protein control)

  • Dose-response series (1-100 nM rSPINK9)

  • Positive control (EGF or another known motility factor)

• Inhibition controls:

  • EGFR inhibitor (e.g., AG1478 or cetuximab)

  • Metalloprotease inhibitor (e.g., GM6001)

  • Purinergic receptor antagonists

  • Specific siRNAs targeting pathway components

• Antibody specificity controls:

  • Pre-immune serum or isotype control antibody

  • Antibody pre-absorbed with recombinant SPINK9

  • Secondary antibody-only control

• Migration assay controls:

  • Fixed timepoint documentation (0h, 6h, 12h, 24h)

  • Multiple field quantification (minimum 5 fields per condition)

  • Duplicate or triplicate experimental replicates

  • Cell proliferation control (mitomycin C treatment)

• Analysis controls:

  • Blinded quantification methods

  • Automated and manual measurement comparison

  • Statistical validation with appropriate tests

How can I optimize Western blot conditions for detecting low levels of SPINK9 protein?

To optimize Western blot detection of low-abundance SPINK9 protein:

• Sample preparation optimization:

  • Enrich SPINK9 via immunoprecipitation before Western blotting

  • Use specialized extraction buffers for secreted proteins

  • Concentrate samples using TCA precipitation or similar methods

  • For skin samples, separate epidermis from dermis to concentrate signal

• Gel and transfer parameters:

  • Use high percentage gels (15-20%) for better resolution of low MW proteins

  • Optimize transfer conditions for small proteins:

    • Lower methanol concentration (10-15%)

    • Shorter transfer time (30-60 minutes)

    • Lower voltage transfer (30V overnight)

  • Consider semi-dry transfer systems for efficient small protein transfer

• Blocking and antibody conditions:

  • Test different blocking agents (BSA vs. milk protein)

  • Extended primary antibody incubation (overnight at 4°C)

  • Higher primary antibody concentration (1:200-1:500)

  • Use high-sensitivity detection systems (enhanced chemiluminescence)

• Signal enhancement strategies:

  • Use signal enhancers compatible with your detection system

  • Consider biotin-streptavidin amplification

  • Explore femto-sensitivity substrates for chemiluminescence

  • Use high-sensitivity fluorescent secondary antibodies

• Exposure optimization:

  • Multiple exposure times (10 seconds to 5 minutes)

  • Digital acquisition with cumulative exposure capability

How should I design experiments to investigate SPINK9's role in purinergic receptor signaling?

To effectively investigate SPINK9's role in purinergic receptor signaling:

• Receptor profiling:

  • Characterize purinergic receptor expression in your experimental system

  • Use qRT-PCR to profile P2X and P2Y receptor subtypes

  • Validate protein expression by Western blotting or flow cytometry

• Antagonist studies:

  • Design a comprehensive antagonist panel:

  Antagonist	Target	Concentration Range	Pre-incubation Time
  Oxidized ATP	P2X7	100-300 μM	1-2 hours
  PPADS	P2X/P2Y	10-100 μM	30 minutes
  Suramin	Broad P2	10-300 μM	30 minutes
  MRS2500	P2Y1	0.1-10 μM	30 minutes
  AR-C118925XX	P2Y2	1-10 μM	30 minutes

• Calcium signaling assays:

  • Monitor intracellular calcium using fluorescent indicators (Fluo-4, Fura-2)

  • Compare calcium responses to SPINK9 versus ATP (purinergic control)

  • Perform real-time imaging to capture temporal dynamics

• Receptor knockdown validation:

  • Use siRNA to target specific purinergic receptor subtypes

  • Verify knockdown efficiency (>70%) before functional studies

  • Test SPINK9 responses in knockdown cells

• Downstream signaling analysis:

  • Monitor PLC activation and IP3 production

  • Assess ADAM17/10 activation following SPINK9 stimulation

  • Determine if calcium chelators block SPINK9's effects

• Co-immunoprecipitation studies:

  • Investigate potential physical associations between SPINK9 and purinergic receptors

  • Use crosslinking approaches for transient interactions

What are the key considerations when comparing SPINK9 expression across different skin conditions or disease states?

When comparing SPINK9 expression across different skin conditions:

• Sample standardization:

  • Match samples for anatomical location (palmar-plantar vs. other sites)

  • Control for age, gender, and other demographic variables

  • Standardize sample collection, processing, and storage

  • Include site-matched controls for each disease sample

• Quantification methods:

  • Implement multiple measurement approaches:

  Method	Measurement	Normalization
  IHC	H-score or percentage positive cells	Tissue area or cell count
  qRT-PCR	Ct values	Multiple reference genes (GAPDH, ACTB, RPLP0)
  Western blot	Band intensity	Total protein (Ponceau) or housekeeping proteins
  ELISA	Protein concentration	Total protein concentration

• Statistical considerations:

  • Determine appropriate sample size through power analysis

  • Use paired statistical tests when comparing diseased and healthy skin from the same individuals

  • Apply multiple testing correction for large-scale comparisons

  • Consider non-parametric tests for non-normally distributed data

• Result validation:

  • Confirm findings with at least two independent methods

  • Verify protein expression correlates with mRNA levels

  • Include positive and negative tissue controls in each experimental batch

  • Consider blinded analysis to prevent bias

How can I determine if my experimental conditions affect SPINK9 antibody epitope accessibility?

To systematically evaluate epitope accessibility under different experimental conditions:

• Epitope mapping analysis:

  • Determine which region of SPINK9 your antibody recognizes:

    • AA 20-86 region antibodies (common commercial type)

    • Middle region antibodies

    • C-terminal region antibodies

  • Consult immunogen sequence information provided by manufacturer

• Structural considerations:

  • SPINK9 contains disulfide bonds that may affect epitope exposure

  • Compare reducing vs. non-reducing conditions in Western blots

  • Evaluate native vs. denatured protein detection efficiency

• Fixation impact assessment:

  • Test a matrix of fixation methods:

  Fixation	Duration	Temperature	Impact on Epitope
  4% PFA	10-30 min	RT	Document effect
  Methanol	5-15 min	-20°C	Document effect
  Acetone	2-10 min	-20°C	Document effect
  Combined	Various	Various	Document effect

• Antigen retrieval optimization:

  • Compare heat-induced vs. enzymatic retrieval methods

  • Test pH gradients (pH 6.0 vs. pH 9.0 buffers)

  • Evaluate microwave vs. pressure cooker heating methods

• Blocking condition effects:

  • Compare different blocking agents (BSA, normal serum, commercial blockers)

  • Test whether blocking affects epitope accessibility

• Post-translational modification considerations:

  • Determine if glycosylation or other modifications mask epitopes

  • Consider enzymatic treatments to remove modifications if necessary

What methodological approaches should be used to study SPINK9's interaction with kallikrein-related peptidase 5 (KLK5)?

To comprehensively characterize SPINK9-KLK5 interactions:

• In vitro binding assays:

  • ELISA-based binding assays with purified recombinant proteins

  • Surface Plasmon Resonance (SPR) to determine binding kinetics (kon, koff, KD)

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters

  • Co-immunoprecipitation from cell lysates or conditioned media

• Enzymatic inhibition characterization:

  • Determine inhibition constant (Ki) using purified recombinant proteins

  • Use fluorogenic substrates to monitor KLK5 activity

  • Perform Lineweaver-Burk analysis to determine mode of inhibition

  • Develop a competitive binding assay with known KLK5 substrates

• Structural studies:

  • X-ray crystallography of SPINK9-KLK5 complex

  • NMR spectroscopy for solution-phase interaction analysis

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Molecular modeling and docking simulations

• Cell-based functional assays:

  • Co-expression studies in relevant cell models

  • Proximity ligation assay (PLA) to detect in situ interactions

  • FRET/BRET-based interaction assays

  • Functional readouts of KLK5 activity in presence/absence of SPINK9

• Domain mapping:

  • Generate truncated or point-mutated versions of both proteins

  • Test each variant in binding and inhibition assays

  • Identify critical residues for interaction

How can I design multiplex imaging experiments to study SPINK9 co-localization with other skin barrier proteins?

For effective multiplex imaging of SPINK9 with other skin barrier proteins:

• Antibody panel design:

  • Carefully select primary antibodies from different host species:

  Target	Host Species	Clonality	Notes
  SPINK9	Rabbit	Polyclonal	Validated in IHC/IF
  KLK5	Mouse/Goat	Monoclonal	Primary interaction partner
  Filaggrin	Mouse/Chicken	Mono/Poly	Differentiation marker
  Loricrin	Goat	Polyclonal	Terminal differentiation
  EGFR	Mouse	Monoclonal	Signaling pathway

• Fluorophore selection:

  • Choose spectrally separated fluorophores to minimize bleed-through

  • Include single-color controls for spectral unmixing

  • Consider brightness, photobleaching resistance, and quantum yield

• Sample preparation optimization:

  • Test different fixation protocols to preserve all antigens

  • Optimize antigen retrieval conditions compatible with all targets

  • Determine ideal blocking conditions to prevent non-specific binding

  • Consider sequential staining for problematic antibody combinations

• Advanced imaging techniques:

  • Confocal microscopy for high-resolution co-localization analysis

  • Super-resolution techniques (STED, STORM, PALM) for nanoscale distribution

  • Spectral imaging with linear unmixing for overlapping fluorophores

  • Live-cell imaging for dynamic studies (if applicable)

• Quantitative analysis methods:

  • Calculate Pearson's or Mander's coefficients for co-localization

  • Perform distance analysis between different protein signals

  • Consider object-based co-localization for clustered proteins

  • Use 3D reconstruction for volumetric co-localization analysis

What experimental design considerations are important when investigating SPINK9's relationship with other antimicrobial peptides in skin immunity?

When investigating SPINK9's relationship with other antimicrobial peptides (AMPs):

• Experimental model selection:

  • Primary human keratinocytes (most physiologically relevant)

  • Reconstituted human epidermis (3D model)

  • Skin explant cultures (maintains tissue architecture)

  • Mouse models (consider species differences in AMP expression)

• Co-expression analysis:

  • Comparative expression profiling of SPINK9 and other AMPs:

  Analysis Type	Method	Sample Type	Data Output
  mRNA	RNAseq/qPCR	Tissues/Cells	Expression correlation
  Protein	Multiplex IHC	Tissue sections	Spatial relationships
  Secretome	Mass spec	Conditioned media	Secretion patterns

• Functional interaction studies:

  • Test for synergistic, additive, or antagonistic effects:

    • Antimicrobial activity assays with combined peptides

    • Cell migration/proliferation with peptide combinations

    • Inflammatory response modulation (cytokine production)

    • Barrier function assessments (TEER measurements)

• Stimulation conditions:

  • Compare different induction stimuli:

    • Microbial components (LPS, peptidoglycan, zymosan)

    • Barrier disruption models (tape stripping, detergent)

    • Inflammatory cytokines (IL-17, IL-22, TNF-α)

    • Disease-relevant conditions (psoriasis-like, atopic-like)

• Temporal dynamics:

  • Time-course experiments to determine expression sequence

  • Pulse-chase studies to assess protein turnover

  • Real-time monitoring of induction and secretion

• Mechanistic investigations:

  • Promoter analysis for common regulatory elements

  • Signal transduction pathway inhibition studies

  • ChIP-seq for shared transcription factor binding

  • Co-immunoprecipitation for protein complex formation