CLDN17 Antibody

What is CLDN17 and where is it primarily expressed in mammalian tissues?

CLDN17 is a member of the claudin family of tight junction proteins that have isoform-specific roles in blood-tissue barrier regulation. It is a putative anion pore-forming claudin based on its structural characterization and is assumed to regulate anion balance across blood-tissue barriers . CLDN17 is predominantly expressed in the kidneys, particularly in the proximal segments of the nephron, with a gradual decrease in expression from the proximal tubule (PCT) downstream to the distal convoluted tubule (DCT) . It is also expressed in the brain and forms distinct anion-selective channels that are structurally and functionally different from paracellular cation channels .

How does CLDN17 differ functionally from other claudin family proteins?

Unlike many claudins that primarily seal the paracellular barrier, CLDN17 forms paracellular channels with distinct anion selectivity. When overexpressed in MDCK C7 cell layers, CLDN17 causes a threefold increase in paracellular anion permeability and can switch these cells from cation- to anion-selective . This makes CLDN17 unique among claudins, as few claudins with general and clear-cut anion selectivity have been described. The gene coding for CLDN17 is clustered with the CLDN8 gene on chromosome 21q22.11, and both claudins share high sequence similarity. Interestingly, CLDN17 may replace CLDN8 to recruit occludin (OCLN) in tissues where CLDN8 is absent, suggesting complementary roles between these two claudins .

What are the key considerations when selecting a CLDN17 antibody for research purposes?

When selecting a CLDN17 antibody, researchers should consider:

• Epitope specificity: Choose antibodies targeting specific amino acid regions like AA 29-81 or AA 103-152 depending on your research question

• Host species: Most available antibodies are raised in rabbits as polyclonal antibodies

• Cross-reactivity: Verify the antibody's species reactivity (human, mouse, rat, etc.) to ensure compatibility with your experimental model

• Application compatibility: Ensure the antibody is validated for your specific application (WB, IHC, IF, ELISA, etc.)

• Clonality: Most commercially available CLDN17 antibodies are polyclonal, which provides good sensitivity but may have batch-to-batch variation

What are the optimal protocols for using CLDN17 antibodies in Western blotting?

For Western blotting with CLDN17 antibodies, follow these methodological recommendations:

• Sample preparation: Extract total protein from tissues (especially kidney) or cell lines known to express CLDN17 using an appropriate lysis buffer containing protease inhibitors

• Protein separation: Use SDS-PAGE (10-12% gels) to separate proteins, with particular attention to loading controls

• Transfer conditions: Transfer proteins to PVDF or nitrocellulose membranes (0.2 μm pore size recommended for smaller proteins like claudins)

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

• Primary antibody incubation: Dilute CLDN17 antibody (typically 1:500 to 1:1000) in blocking buffer and incubate overnight at 4°C

• Detection: Use appropriate secondary antibodies conjugated to HRP followed by ECL detection

When troubleshooting, consider that CLDN17 has a molecular weight of approximately 23-25 kDa, and non-specific bands may appear due to cross-reactivity with other claudin family members given their sequence similarities.

How should CLDN17 antibodies be applied in immunohistochemistry studies of kidney tissue?

For optimal IHC results with CLDN17 antibodies in kidney tissue:

• Tissue preparation: Use formaldehyde-fixed, paraffin-embedded tissue sections (5-7 μm thickness)

• Antigen retrieval: Perform heat-induced antigen retrieval by boiling sections in 10 mM sodium citrate buffer (pH 6.0) for 20 minutes

• Permeabilization: Treat sections with PBS containing 0.5% (v/v) Triton X-100

• Blocking:

  • First block with 5% goat or rabbit serum (depending on secondary antibody species) in PBS containing magnesium and calcium for 30 minutes

  • Follow with 30 minutes in immunofluorescence buffer (0.1% Triton-X 100, 0.15 M NaCl, 5 mM EDTA, 20 mM HEPES, pH 7.5)

• Antibody dilution: Dilute primary CLDN17 antibodies in immunofluorescence buffer (typically 1:25 to 1:100 depending on the antibody)

• Visualization: For co-localization studies, consider dual immunostaining with markers of specific nephron segments (e.g., NKCC2 for thick ascending limb)

What approaches can be used to verify the specificity of CLDN17 antibody signals?

To verify CLDN17 antibody specificity:

• Positive and negative tissue controls: Use kidney tissue (high expression) as positive control and tissues known not to express CLDN17 as negative control

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to block specific binding

• Genetic models: Use samples from CLDN17 knockout or knockdown models as negative controls

• Multiple antibody validation: Use antibodies targeting different epitopes of CLDN17 and compare staining patterns

• siRNA knockdown: In cell culture, perform siRNA-mediated knockdown of CLDN17 using validated sequences (e.g., 5′-AACATTATTGTCTTTGAAAGG-3′, 5′-AAGTTCTACAGTTCTATGCTG-3′, 5′-AATATCATCATCAGGGACTTC-3′)

• RT-qPCR correlation: Correlate protein expression with mRNA levels using primers (e.g., 5′-tctccctccggtactggaag-3' and 5′-gctcctccaagttctcgctt-3')

How can CLDN17 antibodies be employed to investigate kidney pathophysiology?

CLDN17 antibodies can be powerful tools for investigating kidney pathophysiology:

• Expression pattern analysis: Map CLDN17 distribution across nephron segments under normal and pathological conditions using immunofluorescence or immunohistochemistry

• Protein-protein interaction studies: Use co-immunoprecipitation with CLDN17 antibodies to identify interaction partners in tight junctions

• Pathological alterations: Assess changes in CLDN17 expression in models of:

  • Electrolyte imbalance disorders

  • Oxidative stress-induced kidney injury

  • Inflammation-related kidney diseases

Research has shown that CLDN17 deficiency in mice results in electrolyte imbalance, oxidative stress, and kidney injury. RNA-sequencing and Ingenuity pathway analysis revealed hyperactivation of signaling pathways and downregulation of SOD1 expression in kidneys associated with inflammation and reactive oxygen species generation . CLDN17 antibodies can help track these changes at the protein level.

What are the methodological considerations when using CLDN17 antibodies in knockout or knockdown studies?

When using CLDN17 antibodies in genetic manipulation studies:

• Knockout verification: Use CLDN17 antibodies to confirm complete protein elimination in knockout models

• Knockdown efficiency assessment: Quantify the reduction in CLDN17 protein levels following siRNA treatment using Western blotting

• Compensatory changes: Investigate potential upregulation of other claudins (particularly CLDN8) in response to CLDN17 depletion

• Phenotype correlation: Correlate antibody-detected protein levels with functional parameters (e.g., electrolyte levels, markers of oxidative stress)

For CLDN17 knockdown, researchers have successfully used siRNA sequences targeting porcine CLDN17: 5′-AACATTATTGTCTTTGAAAGG-3′, 5′-AAGTTCTACAGTTCTATGCTG-3′, and 5′-AATATCATCATCAGGGACTTC-3′ with Fugene HD transfection reagent . CRISPR/Cas9 technology has been used to generate CLDN17 knockout mice with gRNA binding/PAM site: 5′-TCGGTTTGGTTGGGACGATTGGG-3′ .

How can CLDN17 antibodies help elucidate the molecular mechanism of anion channel formation?

CLDN17 antibodies can contribute to understanding anion channel formation through:

• Structure-function analysis: Use antibodies to detect expression of CLDN17 mutants (e.g., K65E, K65A mutations) and correlate with functional changes in anion selectivity

• Localization studies: Determine whether mutations affect proper membrane localization of CLDN17 in tight junctions

• Interaction analysis: Identify which domains of CLDN17 interact with scaffold proteins of the tight junction complex

• Electrophysiological correlation: Combine antibody-based detection of CLDN17 with functional measurements of paracellular anion permeability

Overexpression of CLDN17 in MDCK C7 cells increases anion permeability threefold and switches these cells from cation- to anion-selective, while knockdown in LLC-PK1 cells (which naturally express CLDN17) reduces anion permeability .

What are common pitfalls in CLDN17 antibody-based experiments and how can they be addressed?

Common challenges in CLDN17 antibody experiments include:

• Cross-reactivity with other claudins: Due to sequence similarity between claudin family members, particularly between CLDN17 and CLDN8. Solution: Use antibodies targeting unique epitopes and validate with positive and negative controls

• Low signal in kidney tissue: Solution: Optimize antigen retrieval (10 mM sodium citrate buffer, pH 6.0, 20 min boiling) and use signal amplification systems

• Variability between antibody lots: Solution: Validate each new lot against a reference sample

• Developmental expression differences: CLDN17-/- mice show delayed growth in newborn pups , suggesting developmental regulation. Solution: Age-match experimental animals carefully

• Species differences: Consider species-specific differences in CLDN17 expression and antibody reactivity when translating between animal models

How should researchers interpret CLDN17 antibody signals in the context of kidney function studies?

For proper interpretation of CLDN17 antibody signals in kidney studies:

• Nephron segment identification: Always co-stain with segment-specific markers to identify the precise tubular location of CLDN17 signals

• Quantification approach: Use digital image analysis with appropriate controls for accurate quantification of signal intensity

• Functional correlation: Correlate CLDN17 protein levels with:

  • Serum electrolyte measurements (especially anions)

  • Markers of oxidative stress (CLDN17-/- mice show increased ROS levels)

  • Inflammatory markers (CLDN17 deficiency is associated with inflammatory pathway activation)

• Context of other claudins: Interpret CLDN17 expression patterns in the context of other claudins, as CLDN17 deficiency affects expression of CLDN3-7, CLDN9, and CLDN19

What controls are essential when detecting CLDN17 in experimental systems?

Essential controls for CLDN17 detection include:

• Positive tissue control: Kidney proximal tubule sections known to express CLDN17

• Negative tissue control: Tissues known not to express CLDN17

• Antibody controls:

  • Primary antibody omission

  • Isotype control

  • Peptide competition/blocking control

• Genetic controls:

  • CLDN17 knockdown or knockout samples

  • Overexpression systems with verified CLDN17 expression

• Loading/housekeeping controls: For Western blots, include appropriate loading controls (β-actin has been used successfully)

• mRNA correlation: Validate protein expression patterns with RT-qPCR using validated primers (5′-tctccctccggtactggaag-3' forward and 5′-gctcctccaagttctcgctt-3' reverse)

How might CLDN17 antibodies contribute to understanding disease mechanisms beyond the kidney?

While CLDN17 is primarily studied in the kidney, antibodies against this protein could help explore its role in:

• Brain barrier function: CLDN17 is expressed in the brain , suggesting potential roles in blood-brain barrier regulation

• Cancer research: Some studies have investigated CLDN17 in cancer cells in vitro

• Developmental biology: Given the growth delays observed in CLDN17-/- pups , antibodies could help track CLDN17 expression during development

• Comparative physiology: Studying CLDN17 expression across species may reveal evolutionary adaptations in tight junction regulation

What novel methodological approaches might enhance CLDN17 antibody-based research?

Emerging methods to consider for CLDN17 antibody applications:

• Super-resolution microscopy: To better visualize CLDN17 organization within tight junction complexes

• Proximity ligation assays: To detect and quantify interactions between CLDN17 and other tight junction proteins

• Live-cell imaging: Using fluorescently-tagged antibody fragments to track CLDN17 dynamics

• Mass spectrometry immunoprecipitation: To identify the complete interactome of CLDN17 in different physiological states

• Single-cell analysis: Combining CLDN17 antibody staining with single-cell transcriptomics to correlate protein expression with gene expression profiles