SPINK6 Antibody

What is the optimal application range for SPINK6 antibodies in tissue analysis?

SPINK6 antibodies demonstrate versatility across several applications, with particular strength in immunohistochemistry (IHC) and Western blotting. For IHC applications, most commercial antibodies perform optimally at dilutions between 1:50-1:300, with rabbit polyclonal antibodies showing strong reactivity in human tissue samples, particularly in epithelial cells of the respiratory tract and skin . For Western blotting, antibodies such as ab201319 have demonstrated effectiveness across human, mouse, and rat samples, with an expected band size of approximately 9 kDa .

When analyzing SPINK6 expression in lung tissues, researchers should be aware that optimal staining may require antigen retrieval techniques and careful titration of antibody concentrations, as demonstrated in studies of human lung carcinoma tissues .

How should researchers interpret SPINK6 expression data in human tissues?

When interpreting SPINK6 expression, researchers should establish appropriate scoring systems. In published studies of nasopharyngeal carcinoma, investigators used median scores (e.g., score = 3) as cut-off values to divide patients into high (>median) and low (≤median) SPINK6 expression groups . This approach provided prognostic stratification that correlated with clinical outcomes.

For quantitative analysis, Image-Pro Plus 6.0 or similar software can be employed for relative quantification of expression gradation . When examining SPINK6 expression in airway tissues, immunofluorescence staining reveals that over 30% of cells in differentiated airway organoids express SPINK6, with a large proportion co-expressing HAT (Human Airway Trypsin-like protease) .

How can researchers optimize co-immunoprecipitation protocols to study SPINK6 interactions with target proteases?

For effective co-immunoprecipitation (Co-IP) of SPINK6 with potential binding partners such as EGFR:

• Preparation of recombinant proteins: Express tagged versions of both proteins (e.g., 3× Flag-tagged EGFR and Myc-His–tagged SPINK6) in cell-free expression systems to study direct interactions .

• Optimization of binding conditions:

  • Mix the recombinant proteins and incubate at 4°C with inversion overnight

  • Add appropriate affinity gel (e.g., anti-FLAG M2 Affinity Gel for FLAG-tagged proteins)

  • Continue incubation for 4 hours at 4°C

  • Collect precipitates by centrifugation and analyze by Western blotting

• Analysis of endogenous interactions:

  • Prepare cell lysates in weak RIPA buffer

  • Incubate with specific antibodies (e.g., anti-EGFR or anti-His-tag) for 12 hours at 4°C

  • Add Protein A/G PLUS-Agarose for an additional 4 hours at 4°C

  • Wash precipitates thoroughly before Western blot analysis

For pull-down assays specifically, researchers have successfully used 2 μg/mL Fc-tagged human EGFR protein mixed with 2 μg/mL recombinant SPINK6 in PBS buffer, demonstrating that SPINK6 can directly bind to the extracellular domain of EGFR through subdomains 1 and 3 .

What are the methodological considerations for studying SPINK6 inhibition of serine proteases in respiratory infections?

When investigating SPINK6's role in respiratory infections, particularly influenza:

• Cell-free protease activity assays: Use fluorogenic substrates of serine proteases to measure SPINK6 inhibition of proteases like TPCK trypsin, HAT, and KLK5. Compare wild-type SPINK6 (wtSPINK6) with mutant versions (mutSPINK6) carrying loss-of-function mutations in the protease inhibition domain .

• Viral replication models:

  • Transfect cells (e.g., BHK21 or A549) with proteases (HAT, TMPRSS2, or Matriptase) with or without SPINK6

  • Inoculate with appropriate influenza virus strains (H7N9/ah or H1N1/pdm)

  • Harvest cell-free culture media at 24h post-infection

  • Quantify viral growth using plaque assays

• Airway organoid models:

  • Establish differentiated human airway organoids from primary lung tissues

  • Verify SPINK6 expression by immunofluorescence staining and flow cytometry

  • Monitor expression changes of proteases (HAT, KLK5, TMPRSS2) and SPINK6 upon viral infection

  • Implement gene silencing or overexpression of SPINK6 to assess functional impact

How can researchers effectively validate SPINK6 antibody specificity for their experimental systems?

To ensure antibody specificity:

• Peptide competition assays: Pre-incubate the antibody with immunizing peptide before application in IHC or Western blotting. As demonstrated with ab110830, staining of human lung carcinoma tissue is abolished in the presence of the immunizing peptide, confirming specificity .

• Multi-antibody validation: Compare results from multiple SPINK6 antibodies targeting different epitopes (e.g., ab110830 targeting a synthetic peptide vs. ab201319 targeting amino acids 1 to C-terminus) .

• Cross-species reactivity testing: Validate antibody performance across different species when relevant. For example, ab201319 shows reactivity with human, mouse (Raw264.7), and rat (H9C2) cell lysates in Western blotting .

• Negative controls: Include appropriate isotype controls such as Rabbit IgG (A82272 or A17360) when using rabbit polyclonal SPINK6 antibodies .

What are optimal experimental designs for investigating SPINK6's role in cancer progression?

Based on studies of nasopharyngeal carcinoma, researchers should consider:

• Expression analysis in patient cohorts:

  • Analyze SPINK6 expression in tumor samples using IHC (recommended dilution 1:50)

  • Score intensity independently by two pathologists to ensure reliability

  • Correlate expression with clinical parameters using appropriate statistical methods (Spearman correlation, Kaplan-Meier survival analysis, Cox regression)

• Functional investigation in vitro:

  • Generate stable SPINK6-overexpressing and SPINK6-silenced cell lines

  • Assess effects on migration and invasion using transwell assays

  • Investigate epithelial-mesenchymal transition markers (e.g., vimentin) by Western blotting and IHC

  • Examine activation of signaling pathways (EGFR, AKT) using phospho-specific antibodies

• In vivo metastasis models:

  • Establish animal models to assess lymph node metastasis and liver metastasis

  • Test intervention strategies, such as EGFR inhibition with erlotinib, in SPINK6-driven metastasis

  • Use both gain- and loss-of-function approaches to confirm SPINK6's role

How should researchers approach SPINK6 antibody selection for multi-parameter flow cytometry experiments?

For flow cytometry applications:

• Antibody selection criteria:

  • Choose antibodies validated for flow cytometry applications

  • Ensure the selected clone doesn't compete with antibodies against potential co-markers

  • Select appropriate fluorophore conjugates based on the cytometer configuration and panel design

• Panel design for airway organoid analysis:

  • Include markers for SPINK6 and relevant proteases (e.g., HAT, KLK5)

  • Add cell type-specific markers to identify epithelial subpopulations

  • Include viability dye to exclude dead cells

• Titration and compensation:

  • Titrate antibodies to determine optimal concentration

  • Perform proper compensation controls, especially important when studying co-expression

  • Include FMO (Fluorescence Minus One) controls to set accurate gates

In published research, flow cytometry analysis has shown that over 30% of cells in differentiated airway organoids express SPINK6, with significant co-expression with HAT . This approach allows for quantitative assessment of SPINK6 expression in specific cell populations.

How should researchers resolve contradictions in SPINK6 expression data across different tissue types?

When encountering discrepancies in SPINK6 expression across tissues:

• Methodological considerations:

  • Compare antibody clones, epitopes, and applications used

  • Assess tissue preservation methods and antigen retrieval protocols

  • Consider sensitivity differences between detection methods (IHC vs. Western blot vs. qPCR)

• Biological interpretation:

  • Recognize that SPINK6 expression may be context-dependent and regulated by tissue-specific factors

  • Consider disease state and inflammatory conditions, as SPINK6 is upregulated in response to infections

  • Analyze expression in relation to target proteases (KLKs) in each tissue type

• Reconciliation strategies:

  • Employ multiple detection methods on the same samples

  • Validate with recombinant protein controls

  • Consider single-cell analysis to identify cell type-specific expression patterns

Research has demonstrated that SPINK6 expression varies significantly between normal tissues and cancer samples, and is upregulated in response to influenza infection in airway organoids , suggesting context-dependent regulation.

What statistical approaches are most appropriate for analyzing SPINK6 expression in relation to clinical outcomes?

For robust statistical analysis:

• Categorical analysis:

  • Divide patients into expression groups using appropriate cut-off values (e.g., median score)

  • Apply χ² test for association with categorical clinical variables

  • Use Kaplan-Meier method with log-rank analysis for survival outcomes

• Continuous variable approaches:

  • Employ Spearman correlation analysis for relationships between SPINK6 and other markers (e.g., pEGFR, vimentin)

  • Use unpaired Student's t-test for comparing expression levels between groups

  • Apply Cox regression model for multivariate survival analysis

• Multivariate analysis:

  • Include relevant clinical covariates (stage, age, treatment)

  • Test for independent prognostic value of SPINK6 expression

  • Consider interaction terms between SPINK6 and related biomarkers

What are the key technical considerations when using SPINK6 antibodies for immunohistochemistry in formalin-fixed tissues?

For optimal IHC results in formalin-fixed, paraffin-embedded (FFPE) tissues:

• Antigen retrieval optimization:

  • Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

  • Optimize pH conditions for epitope exposure

  • Adjust retrieval duration based on fixation time of tissues

• Antibody dilution and incubation:

  • Start with manufacturer-recommended dilutions (e.g., 1:50-1:300)

  • Perform antibody titration to determine optimal concentration

  • Consider extended primary antibody incubation at 4°C overnight

• Detection system selection:

  • Choose appropriate secondary antibodies (e.g., Goat Anti-Rabbit IgG H&L)

  • Select detection method based on sensitivity requirements (HRP, AP, FITC, or Biotin conjugates)

  • Include proper controls (isotype, peptide competition, known positive tissues)

Commercial antibodies such as ab110830 have been successfully used for IHC-P applications in human lung carcinoma tissues, demonstrating specific staining that is abolished by peptide competition .

How can researchers effectively combine SPINK6 detection with functional protease activity assays?

To integrate expression and functional analysis:

• Sequential tissue section analysis:

  • Perform IHC for SPINK6 on one section

  • Use adjacent sections for in situ zymography to detect protease activity

  • Correlate patterns of inhibitor expression with substrate cleavage

• Cell-based activity assays:

  • Measure protease activity using fluorogenic substrates in SPINK6-expressing and control cells

  • Assess dose-dependent inhibition using recombinant SPINK6 protein

  • Compare wild-type SPINK6 with mutant versions lacking inhibitory activity

• Combined detection strategies:

  • Implement dual immunofluorescence for SPINK6 and target proteases

  • Correlate with functional readouts such as viral activation in infection models

  • Use protein-protein interaction assays (Co-IP, pull-down) to confirm direct inhibition

Research has demonstrated that SPINK6 specifically suppresses the proteolytic activity of HAT and KLK5, affecting processes like HA cleavage of influenza viruses, which can be measured through both molecular and functional assays .