SPINK6 Antibody, FITC conjugated

What is SPINK6 and what is its physiological role?

SPINK6 is a serine protease inhibitor belonging to the Kazal-type family, possessing only one typical Kazal domain (unlike LEKTI but similar to LEKTI-2). It functions as a selective inhibitor of kallikreins in human skin and potentially other tissues . SPINK6 efficiently inhibits KLK4, KLK5, KLK6, KLK7, KLK12, KLK13, and KLK14, but doesn't inhibit KLK8 . This selective inhibition pattern suggests SPINK6 plays a specialized role in regulating protease activity in epithelial tissues.

In skin physiology, SPINK6 is expressed in the stratum granulosum and skin appendages including sebaceous glands and sweat glands . It modulates the activity of kallikreins that are central to skin desquamation processes. SPINK6 has been shown to inhibit desquamation of human plantar callus in ex vivo models, indicating its role in regulating skin barrier function . Additionally, decreased SPINK6 expression has been observed in lesions of atopic dermatitis, suggesting a potential role in skin pathologies .

Recent research has also identified SPINK6 as an inhibitor of human airway serine proteases that can restrict influenza virus activation, indicating broader physiological significance beyond skin biology .

How does SPINK6 compare to other SPINK family members in structure and function?

SPINK6 differs structurally from some other SPINK family members. While LEKTI (encoded by SPINK5) contains multiple Kazal domains, SPINK6 possesses only one typical Kazal domain, similar to LEKTI-2 (encoded by SPINK9) . This structural difference may explain the different inhibitory profiles observed among SPINK proteins.

Functionally, SPINK6 shows selective inhibition of specific kallikreins with varying affinities. Research has demonstrated that SPINK6 inhibits KLK5, KLK7, and KLK14 with apparent Ki values of 1.33, 1070, and 0.5 nM, respectively . This selective inhibition differs from the broader inhibitory profiles of some other SPINK family members.

What are the technical specifications of commercially available SPINK6 Antibody, FITC conjugated?

Commercial SPINK6 Antibody, FITC conjugated is typically a polyclonal antibody raised in rabbit against recombinant human SPINK6 protein (residues 24-80AA) . The antibody undergoes protein G purification with purity typically exceeding 95% . It's conjugated to FITC (Fluorescein isothiocyanate) to facilitate direct fluorescence-based detection without requiring secondary antibodies.

The antibody is reactive against human SPINK6 and is typically supplied in a buffer containing preservatives like 0.03% Proclin 300 and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) . Storage recommendations generally include keeping the antibody at -20°C or -80°C, avoiding repeated freeze-thaw cycles.

What experimental applications is SPINK6 Antibody, FITC conjugated suitable for?

SPINK6 Antibody, FITC conjugated is well-suited for several research applications:

• Flow cytometry - The FITC conjugation allows direct detection of SPINK6-expressing cells in single-cell suspensions.

• Immunofluorescence microscopy - For detecting SPINK6 expression in tissue sections or cultured cells.

• ELISA - For quantitative measurement of SPINK6 in biological samples.

• Immunohistochemistry - With appropriate modifications to detection methods, considering the FITC conjugation.

Published research demonstrates the use of anti-SPINK6 antibodies for detecting expression in skin tissue sections, particularly in the stratum granulosum and skin appendages . Similar applications would be possible with FITC-conjugated versions, with the added benefit of direct fluorescence detection.

What are the optimal protocols for immunohistochemistry using SPINK6 Antibody?

Based on published methodologies for SPINK6 immunodetection:

For paraffin-embedded tissue sections:

• Deparaffinize and rehydrate tissue sections following standard protocols.

• Perform antigen retrieval (heat-induced epitope retrieval in citrate buffer pH 6.0 is often effective).

• Block endogenous peroxidase with H₂O₂ and perform protein blocking.

• For non-FITC conjugated antibodies, incubate with primary anti-SPINK6 antibody (optimal concentration approximately 10 ng/μl) followed by appropriate secondary antibody .

• For FITC-conjugated antibodies, direct incubation followed by washing is sufficient, with no secondary antibody needed.

• For chromogenic detection (if not utilizing fluorescence), additional steps with appropriate detection systems would be needed.

• Counterstain, dehydrate, and mount as appropriate for visualization method.

Critical controls should include:

• Blocking controls using recombinant SPINK6 peptide to verify antibody specificity

• Negative controls using preimmune sera

• Positive controls using tissues known to express SPINK6 (e.g., stratum granulosum of healthy human skin)

How should researchers optimize SPINK6 detection in airway organoid models?

When studying SPINK6 in airway organoid models, particularly in the context of viral infections like influenza:

• Differentiated 2D airway organoids have demonstrated SPINK6 expression in epithelial cells, with approximately 30% of cells expressing SPINK6 .

• Optimize fixation protocols carefully - paraformaldehyde fixation (4%, 15-20 minutes) is typically suitable for maintaining both structure and epitope accessibility.

• Consider dual staining with markers of airway epithelial cell types and SPINK6 to identify specific cell populations expressing the protein.

• When examining SPINK6 expression in response to viral infection, establish appropriate time points post-infection, as SPINK6 levels may change dynamically during infection progression.

• Flow cytometry analysis can be employed to quantify the percentage of SPINK6-positive cells in organoid cultures, particularly using FITC-conjugated antibodies .

• Consider examining co-expression with proteases like HAT, as research has shown HAT and SPINK6 co-expression in airway epithelial cells .

How can SPINK6 antibodies contribute to research on protease-antiprotease balance in disease models?

SPINK6 antibodies provide critical tools for investigating the protease-antiprotease balance in various disease models:

• In atopic dermatitis research, SPINK6 antibodies can help document the decreased expression observed in lesional skin . This allows correlation of SPINK6 levels with disease severity and other clinical parameters.

• For respiratory virus research, SPINK6 antibodies enable monitoring of this protease inhibitor's expression in response to infection. Research has shown that SPINK6 significantly suppresses HAT and KLK5-mediated viral growth, making it a potential biomarker for susceptibility to respiratory infections .

• Quantitative analysis of SPINK6 levels across different experimental conditions can be achieved through well-designed immunofluorescence or flow cytometry protocols using FITC-conjugated antibodies.

• The inhibitory potency of SPINK6 against specific proteases varies considerably (Ki values ranging from 0.5 nM for KLK14 to 1070 nM for KLK7) . When designing experiments to study this balance, researchers should account for these differential affinities.

The following table summarizes inhibition data for SPINK6 against proteolytic activity:

What experimental approaches can determine the functional significance of SPINK6 in viral infection models?

Several experimental approaches can elucidate SPINK6's functional role in viral infection:

• Gain/loss-of-function studies:

  • Overexpression of SPINK6 in susceptible cell lines followed by viral challenge

  • Knockdown/knockout of SPINK6 using siRNA or CRISPR-Cas9 technologies

  • Application of recombinant SPINK6 protein to cell cultures

• Mechanistic studies:

  • Examine SPINK6 effects on protease-mediated viral activation using fluorogenic substrate assays

  • Assess SPINK6 inhibition of specific proteases (e.g., HAT and KLK5) that are involved in viral activation

  • Monitor cleavage of viral proteins (e.g., hemagglutinin of influenza viruses) in the presence/absence of SPINK6

• Translational approaches:

  • Study SPINK6 expression in human airway organoids, which provide physiologically relevant models

  • Examine effects of SPINK6 administration in mouse models of viral infection, particularly focusing on viral titers and survival outcomes

  • Correlate SPINK6 genetic variants with susceptibility to viral infections in human populations

Research has demonstrated that SPINK6 specifically suppresses HAT and KLK5 activation of influenza viruses and restricts virus maturation and replication. In IAV-infected mice, SPINK6 administration significantly suppresses viral growth and improves survival .

What are common technical challenges when using FITC-conjugated antibodies for SPINK6 detection?

When using SPINK6 Antibody, FITC conjugated, researchers may encounter several technical challenges:

• Photobleaching: FITC is susceptible to photobleaching during extended imaging sessions.

  • Solution: Use anti-fade mounting media, minimize exposure time, and consider using sample preparation methods that enhance signal stability.

• Autofluorescence: Skin tissues often exhibit significant autofluorescence that may interfere with FITC signal.

  • Solution: Include appropriate autofluorescence controls and consider spectral imaging approaches to distinguish true signal from background. Treatment with sodium borohydride or Sudan Black B can reduce autofluorescence in skin sections.

• Weak signal in specific tissues:

  • Solution: Optimize antigen retrieval methods. For SPINK6 detection in skin, published protocols using affinity-purified antibodies at approximately 10 ng/μl concentration have shown good results .

• Cross-reactivity with other SPINK family members:

  • Solution: Perform blocking experiments with recombinant SPINK6 and validation with SPINK6-deficient samples. Specificity testing using recombinant SPINK6 peptide to block the primary antibody has been demonstrated effective .

How should researchers interpret changes in SPINK6 expression across different experimental conditions?

When interpreting SPINK6 expression changes:

• Consider tissue-specific baseline expression:

  • SPINK6 mRNA is detected at low levels in several tissues but is induced during keratinocyte differentiation .

  • In airway organoids, approximately 30% of cells express SPINK6 at baseline, with significant upregulation upon influenza virus infection .

• Account for co-expression patterns:

  • A large proportion of SPINK6-positive cells in airway organoids co-express HAT .

  • This co-expression pattern may indicate coordinated regulation and functional relationships.

• Interpret temporal expression changes carefully:

  • Following influenza infection, significant upregulation of HAT, Furin, and especially KLK5 occurs alongside SPINK6 elevation .

  • This suggests dynamic regulation of the protease-antiprotease balance during infection.

• Consider pathophysiological context:

  • SPINK6 expression is decreased in lesions of atopic dermatitis .

  • Individuals carrying higher SPINK6 expression alleles show protection from human H7N9 influenza infection .