CLDN1 (Ab-210) Antibody

What is the target specificity of CLDN1 (Ab-210) Antibody?

CLDN1 (Ab-210) Antibody is a rabbit polyclonal antibody specifically designed to detect the region around the tyrosine 210 phosphorylation site of human Claudin-1 protein. The antibody is generated using a synthesized non-phosphopeptide derived from human Claudin-1 with the sequence G-K-D-Y(p)-V surrounding the tyrosine 210 site . This antibody detects endogenous levels of total Claudin-1 protein and is affinity-purified from rabbit antiserum using epitope-specific immunogen chromatography .

The antibody demonstrates cross-reactivity with both human and mouse Claudin-1 proteins, making it suitable for comparative studies across these species . Importantly, the antibody is provided at a concentration of 1 mg/ml in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol .

How does CLDN1 (Ab-210) Antibody compare to other Claudin-1 antibodies?

The following table compares various Claudin-1 antibodies and their target regions:

While most Claudin-1 antibodies target larger regions of the protein, the Ab-210 antibody specifically recognizes the tyrosine 210 phosphorylation site, allowing researchers to investigate potential regulatory mechanisms involving this specific amino acid residue .

What are the validated applications for CLDN1 (Ab-210) Antibody?

The CLDN1 (Ab-210) Antibody has been validated for the following applications:

• Western Blotting (WB): Primary application for detecting Claudin-1 protein expression levels in cell and tissue lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of Claudin-1 in various sample types

The antibody has been successfully tested in Western blot analysis of extracts from HeLa cells treated with Hu (2nM, 24 hours), as demonstrated in the validation data provided by manufacturers . While not explicitly validated for immunohistochemistry or immunofluorescence in the Ab-210 variant, other Claudin-1 antibodies in the same product line have been successfully used for these applications .

What controls should be incorporated when using CLDN1 (Ab-210) Antibody in Western blotting experiments?

When designing Western blotting experiments with CLDN1 (Ab-210) Antibody, researchers should include the following controls:

• Positive control: Lysates from tissues or cell lines known to express high levels of Claudin-1 (e.g., liver or kidney tissue extracts)

• Negative control: Either:

  • Samples from Claudin-1 knockout models

  • Samples treated with Claudin-1 siRNA or shRNA (e.g., using claudin-1 siRNA targeting three different claudin-1 regions at 5 nm final concentration)

  • Cell lines with known absence of Claudin-1 expression

• Loading control: Probing for housekeeping proteins such as β-actin, as demonstrated in coimmunoprecipitation studies with Claudin-1

• Antibody specificity control: Pre-incubation of the antibody with the immunizing peptide to confirm signal specificity

• Secondary antibody control: Omitting primary antibody to assess non-specific binding of the secondary antibody

For quantitative Western blot analysis, ImageJ software has been successfully used to analyze Claudin-1 expression levels in multiple studies .

How can CLDN1 (Ab-210) Antibody be used for investigating protein-protein interactions?

CLDN1 (Ab-210) Antibody can be effectively used to study protein-protein interactions involving Claudin-1 through several techniques:

• Co-immunoprecipitation (Co-IP):

  • Use EZviewRed protein G affinity Gel beads for immunoprecipitation

  • Follow with Western blotting using antibodies against potential interaction partners

  • This approach has successfully identified interactions between Claudin-1 and proteins such as ZO-1, claudin-5, and other tight junction components

• Proximity Ligation Assay (PLA):

  • Combines antibody recognition with DNA amplification to detect protein interactions in situ

  • Useful for detecting transient or weak interactions in their native cellular context

• Fluorescence Resonance Energy Transfer (FRET):

  • Requires double transfection with fluorescently tagged proteins (e.g., claudin-1-GFP and potential partner proteins)

  • Has been used to detect homophilic cis- and trans-interactions of Claudin-1 with other tight junction proteins

• Fluorescence Recovery After Photobleaching (FRAP):

  • Useful for studying the dynamics and mobility of Claudin-1 in cell membranes

  • Has been employed to analyze the incorporation of Claudin-1 into tight junction complexes

Research has demonstrated that Claudin-1 interacts with various proteins including nephrin, podocin, and ZO-1, often through both cis- and trans-interactions .

What methodological approaches can be used to study Claudin-1-mediated tight junction formation?

To investigate Claudin-1's role in tight junction formation, researchers can employ several complementary approaches using CLDN1 (Ab-210) Antibody:

• Morphological analysis:

  • Immunofluorescence microscopy to visualize Claudin-1 localization at tight junctions

  • Transmission electron microscopy to examine ultrastructural changes in tight junctions

  • Deep-etching freeze-fracture electron microscopy to resolve ultrastructural changes in tight junction strands

• Functional assays:

  • Trans-epithelial/endothelial electrical resistance (TEER) measurements to assess barrier function

  • Permeability assays using fluorescent tracers of different molecular weights

  • These approaches have been used to demonstrate that Claudin-1 incorporation into tight junctions affects barrier permeability

• Molecular manipulation:

  • Inducible expression systems (e.g., doxycycline-controllable transgene expression)

  • RNA interference using shRNA or siRNA targeting Claudin-1

  • CRISPR/Cas9-mediated genome editing to modify endogenous Claudin-1

• Fusion protein approaches:

  • Creation of fluorescently tagged Claudin-1 constructs (e.g., GFP, mCherry)

  • Full-length open reading frame (ORF) Claudin-1 cloned into vectors such as pmCherry-C1 or pAcGFP-C1

  • These constructs can be used for live-cell imaging of Claudin-1 dynamics

How can CLDN1 (Ab-210) Antibody be utilized in HCV infection research?

The CLDN1 (Ab-210) Antibody can be valuable in hepatitis C virus (HCV) research based on Claudin-1's essential role as an HCV entry factor:

• Detection of Claudin-1 expression in HCV models:

  • Western blotting to quantify Claudin-1 expression levels in HCV-infected cell cultures or tissues

  • Immunohistochemistry to visualize Claudin-1 distribution in liver tissue sections

• Evaluating therapeutic approaches:

  • Assessing changes in Claudin-1 expression following treatment with CLDN1-specific antibodies

  • Monitoring the effectiveness of Claudin-1-targeting therapies in preventing cell-free and cell-cell HCV transmission

• Studying viral entry mechanisms:

  • Investigating Claudin-1's role in both cell-free entry and cell-cell transmission of HCV

  • Analyzing the effects of Claudin-1 antibodies on viral spread in hepatoma cells

Research findings have demonstrated that CLDN1-specific monoclonal antibodies can:

• Prevent de novo HCV infection in a human chimeric mouse model

• Clear chronic HCV infection with genotypes 1b/2a, 2a, and 4

• Exhibit a high genetic barrier to resistance

• Act by interfering with HCV entry rather than replication or assembly/release

How is CLDN1 (Ab-210) Antibody applicable in blood-brain barrier (BBB) research?

CLDN1 (Ab-210) Antibody is particularly useful for studying the pathological role of Claudin-1 in BBB dysfunction:

• Detection of abnormal Claudin-1 expression:

  • While Claudin-1 is rarely expressed at the normal BBB, it becomes highly expressed in leaky brain microvessels after stroke

  • Immunohistochemistry or immunofluorescence to visualize Claudin-1 in brain tissue sections

  • Western blotting of isolated brain microvessels to quantify Claudin-1 expression

• Investigation of tight junction complex dynamics:

  • Studying how Claudin-1 incorporation into the BBB tight junction complex affects other tight junction proteins

  • Analysis of claudin-1/ZO-1 interactions using co-immunoprecipitation

• Therapeutic target evaluation:

  • Assessing the effects of Claudin-1-targeting peptides (e.g., C1C2 peptide) on BBB permeability and recovery

  • Monitoring neurological recovery following stroke when Claudin-1 is targeted

Research has revealed that Claudin-1 is incorporated into BBB tight junctions following stroke, impeding BBB recovery and causing persistent leakiness. Targeting Claudin-1 with specific peptides improves brain endothelial barrier permeability and functional recovery in chronic stroke conditions .

What role does CLDN1 (Ab-210) Antibody play in fibrosis research?

CLDN1 (Ab-210) Antibody can facilitate investigation of Claudin-1's role in fibrotic diseases:

• Detection of Claudin-1 in fibrotic tissues:

  • Western blotting and immunohistochemistry to assess Claudin-1 expression in fibrotic tissues

  • Comparison of expression levels between normal and fibrotic samples

• Evaluation of anti-fibrotic treatments:

  • Monitoring changes in Claudin-1 expression following treatment with anti-CLDN1 monoclonal antibodies

  • Assessing the effectiveness of Claudin-1-targeting approaches in reducing fibrosis

• Mechanistic studies:

  • Investigating how Claudin-1 contributes to excessive deposition of extracellular matrix components

  • Studying the relationship between Claudin-1 expression and fibrogenic pathways

Patent applications have described the use of anti-CLDN1 monoclonal antibodies for the prevention and treatment of fibrotic diseases, particularly pulmonary fibrosis, kidney fibrosis, and skin fibrosis. Research has shown that anti-CLDN1 monoclonal antibodies significantly reduce lung fibrosis in mouse models .

How should researchers interpret variability in Claudin-1 detection across different tissues?

When analyzing Claudin-1 expression patterns using CLDN1 (Ab-210) Antibody, researchers should consider the following factors to interpret variability:

• Tissue-specific expression profiles:

  • Claudin-1 is highly expressed in liver and kidney tissues under normal conditions

  • The protein has a reported length of 211 amino acid residues and a mass of 22.7 kDa

  • Expression levels vary significantly between tissue types and pathological conditions

• Subcellular localization considerations:

  • Under normal conditions, approximately 45% of Claudin-1 is present on the cell membrane

  • Claudin-1 incorporates into tight junction complexes and interacts with ZO-1

  • Changes in subcellular distribution may occur in pathological states

• Pathological alterations:

  • Claudin-1 expression may be induced in tissues where it is normally absent (e.g., in brain endothelial cells after stroke)

  • Expression patterns change during disease progression (e.g., in fibrotic diseases or viral infections)

• Technical considerations:

  • Sample preparation methods can affect epitope accessibility

  • Fixation protocols may influence antibody binding efficiency

  • Protein extraction methods may yield different fractions of membrane-bound proteins

What strategies can address cross-reactivity or non-specific binding issues with CLDN1 (Ab-210) Antibody?

To minimize cross-reactivity and non-specific binding when using CLDN1 (Ab-210) Antibody:

• Optimized blocking protocols:

  • Use 5% BSA or 5% non-fat milk in TBS-T for Western blotting

  • Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Include 0.1-0.3% Triton X-100 for membrane permeabilization in immunohistochemistry

• Antibody validation approaches:

  • Confirm specificity using claudin-1 knockdown or knockout samples

  • Consider using claudin-1 shRNA plasmid DNA (0.5 μg final concentration) or siRNA (5 nm final concentration) targeting different claudin-1 regions

  • Include appropriate negative controls (non-target shRNA or siRNA)

• Optimization of antibody concentration:

  • Perform titration experiments to determine optimal working dilution

  • Starting with manufacturer's recommended dilution (typically 1:1000 for Western blotting)

  • Test multiple concentrations to find balance between signal strength and background

• Complementary detection methods:

  • Validate findings using alternative detection techniques

  • Confirm protein expression with mRNA analysis (RT-PCR, RNA-seq)

  • Use multiple antibodies targeting different epitopes of Claudin-1

How can researchers quantitatively analyze Claudin-1 expression data?

For quantitative analysis of data generated using CLDN1 (Ab-210) Antibody:

• Western blot quantification:

  • Use ImageJ software for densitometric analysis as demonstrated in published protocols

  • Normalize Claudin-1 band intensity to loading controls (β-actin)

  • Include concentration standards if absolute quantification is required

• ELISA-based quantification:

  • Commercial ELISA kits for human Claudin-1 offer detection ranges of 15.6-1000 pg/mL with sensitivity of 3.9 pg/mL

  • Intra-assay precision (CV%<8%) and inter-assay precision (CV%<10%)

  • Sample linearity assessment for serum samples shows 91-101% recovery across dilution ranges

• Immunofluorescence quantification:

  • Use consistent image acquisition parameters

  • Apply background subtraction and thresholding

  • Measure fluorescence intensity per cell or per region of interest

  • Analyze colocalization with other markers using Pearson's or Mander's coefficients

• Statistical analysis considerations:

  • Include sufficient biological and technical replicates

  • Apply appropriate statistical tests (e.g., Mann-Whitney test for non-parametric data)

  • Consider paired analyses for before/after treatment comparisons

How can CLDN1 (Ab-210) Antibody be used to study Claudin-1's role in podocyte pathophysiology?

CLDN1 (Ab-210) Antibody can be instrumental in investigating Claudin-1's pathological role in kidney podocytes:

• Detection of abnormal Claudin-1 expression:

  • Under normal conditions, Claudin-1 is not expressed in mature podocytes

  • Induction of Claudin-1 expression in podocytes causes significant proteinuria

  • Immunohistochemistry and Western blotting to detect Claudin-1 expression in kidney tissues

• Analysis of slit diaphragm-tight junction transition:

  • Investigation of how Claudin-1 induction destabilizes the slit diaphragm protein complex

  • Study of reduced expression and altered localization of nephrin and podocin proteins

  • Deep-etching freeze-fracture electron microscopy to visualize ultrastructural changes

• Protein interaction studies:

  • Analysis of Claudin-1 interactions with nephrin and podocin through cis- and trans-associations

  • Investigation of how these interactions disrupt normal slit diaphragm architecture

Research with transgenic mouse models has demonstrated that inducible expression of Claudin-1 in podocytes causes profound proteinuria by destabilizing the slit diaphragm protein complex. This destabilization involves altered expression and localization of nephrin and podocin, key components of the glomerular filtration barrier .

What methodological approaches can assess therapeutic efficacy of Claudin-1-targeting agents?

CLDN1 (Ab-210) Antibody can support evaluation of therapeutic approaches targeting Claudin-1:

• In vitro efficacy assessment:

  • Cell culture models treated with Claudin-1-targeting agents (antibodies, peptides)

  • Evaluation of barrier function using TEER measurements or permeability assays

  • Application of claudin-1 peptide (C1C2) at 1 μg/ml to cell cultures

• In vivo therapeutic monitoring:

  • Animal models of disease (HCV infection, fibrosis, stroke)

  • Administration of Claudin-1-targeting therapies (e.g., C1C2 peptide at 5 μg/kg)

  • Analysis of tissue samples for changes in Claudin-1 expression and disease markers

• Combination therapy approaches:

  • Assessment of synergistic effects when Claudin-1-targeting therapies are combined with other treatments

  • Investigation of potential resistance mechanisms

• Safety evaluation:

  • Monitoring potential side effects of Claudin-1-targeting therapies

  • Assessment of antibody binding to human and murine CLDN1 to evaluate cross-reactivity

Research has shown promising results with CLDN1-specific monoclonal antibodies in HCV infection models and with claudin-1 peptides in stroke models, demonstrating their potential as therapeutic agents .