CLDND1 Antibody, HRP conjugated

What is CLDND1 and why is it significant in research?

CLDND1 (Claudin domain-containing protein 1, also known as Membrane protein GENX-3745) is a 253 amino acid protein with a calculated molecular weight of 29 kDa that belongs to the claudin family . Claudins are critical components of tight junctions that establish paracellular barriers controlling molecular flow between epithelial cells . CLDND1 is widely distributed in the adult central nervous system, with highest expression in the corpus callosum, caudate nucleus, cerebral cortex, medulla, putamen, spinal cord, substantia nigra, and subthalamic nucleus . Weak expression has also been detected in the adult heart . Recent research has revealed CLDND1's significant role in cancer biology, particularly as a survival factor in basal-like breast cancer cell lines and as a cancer invasion/metastasis suppressor in lung adenocarcinoma . This dual function makes CLDND1 an important target for understanding cancer progression mechanisms and developing potential therapeutic approaches.

What are the key specifications of CLDND1 Antibody, HRP conjugated?

CLDND1 Antibody, HRP conjugated is an immunological reagent designed for sensitive detection of CLDND1 protein in experimental systems. The antibody recognizes the CLDND1 protein (UniProt ID: Q9NY35) . The antibody demonstrates reactivity with human, mouse, and rat samples, making it suitable for cross-species research applications . The horseradish peroxidase (HRP) conjugation typically involves creating a molecular complex between the antibody and HRP enzyme at approximately 1:1 ratio . This conjugation is achieved through specific chemical procedures that maintain both antibody specificity and enzymatic activity . The resulting conjugate enables direct immunocytochemical detection without requiring secondary antibodies, thereby reducing background and enhancing specific signal detection .

How should CLDND1 Antibody, HRP conjugated be stored and handled?

For optimal performance and longevity of CLDND1 Antibody, HRP conjugated, proper storage and handling are essential. The antibody should be stored at -20°C, where it remains stable for approximately one year after shipment . The antibody is typically supplied in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3 to maintain stability . Aliquoting is generally unnecessary for -20°C storage, but for frequent use, dividing into smaller volumes to avoid repeated freeze-thaw cycles is recommended . When working with the antibody, avoid contamination and maintain sterile technique. Prior to use, allow the antibody to equilibrate to room temperature and gently mix by inversion rather than vortexing to prevent denaturation of the antibody or damage to the HRP enzyme. Exposure to strong oxidizing agents should be avoided as they can compromise the HRP activity. For optimal results, follow the manufacturer's specific recommendations for dilution ranges in different applications.

What are the optimal protocols for immunohistochemistry using CLDND1 Antibody, HRP conjugated?

For effective immunohistochemical detection of CLDND1 using HRP-conjugated antibodies, the following optimized protocol is recommended:

Tissue Preparation:

• Fix tissue samples in 4% paraformaldehyde for 24 hours

• Process and embed in paraffin

• Section at 4-6 μm thickness

Staining Protocol:

• Deparaffinize sections through xylene and graded alcohols

• Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) at 95-100°C for 20 minutes

• Allow sections to cool to room temperature (approximately 20 minutes)

• Block endogenous peroxidase activity with 0.3% H₂O₂ in methanol for 30 minutes

• Block non-specific binding with 5% normal serum in PBS containing 0.1% Triton X-100 for 1 hour

• Incubate with CLDND1 Antibody, HRP conjugated at an optimized dilution (typically 1:100-1:500) in blocking buffer for 12-18 hours at 12°C

• Wash thoroughly with PBS containing 0.1% Triton X-100 (3 × 10 minutes)

• Develop signal using DAB substrate

• Counterstain with hematoxylin, dehydrate, and mount

This protocol incorporates findings that the best immunohistochemical results for HRP-conjugated antibodies are obtained using prolonged incubations at 12°C in the presence of 0.1% Triton X-100 . For validation, include appropriate negative controls, including primary antibody preabsorbed with the immunizing peptide, which should obliterate the reaction .

How can I optimize Western blot protocols using CLDND1 Antibody, HRP conjugated?

For optimal Western blot detection of CLDND1 using HRP-conjugated antibodies, follow this detailed protocol:

Sample Preparation:

• Extract total protein from tissues or cells using RIPA buffer supplemented with protease inhibitors

• Determine protein concentration using BCA or Bradford assay

• Prepare samples with reducing Laemmli buffer (20-40 μg protein per lane)

• Heat samples at 95°C for 5 minutes

Electrophoresis and Transfer:

• Resolve proteins on 10-12% SDS-PAGE (CLDND1 has a predicted molecular weight of 29 kDa)

• Transfer to PVDF membrane (0.45 μm pore size) at 100V for 60-90 minutes in cold transfer buffer

Immunodetection:

• Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature

• Incubate with CLDND1 Antibody, HRP conjugated (1:1000-1:5000 dilution) in blocking buffer overnight at 4°C

• Wash thoroughly with TBST (3 × 10 minutes)

• Develop using enhanced chemiluminescence (ECL) substrate

• Image using appropriate detection system

Optimization Considerations:

• For low abundance targets, increase protein loading (up to 60 μg) and extend primary antibody incubation time

• Use fresh transfer buffer and ensure complete transfer by confirming with reversible protein stain

• Validate band specificity using positive control lysates from tissues with known CLDND1 expression (CNS tissues)

• Expected molecular weight should be approximately 29 kDa, but post-translational modifications may alter migration

This protocol leverages the direct HRP conjugation to eliminate secondary antibody incubation steps, resulting in reduced background and faster completion time compared to conventional Western blot protocols.

How can CLDND1 Antibody, HRP conjugated be used to investigate apoptotic pathways in cancer cells?

CLDND1 Antibody, HRP conjugated provides a valuable tool for investigating apoptotic pathways in cancer research, particularly since CLDND1 has been identified as a survival factor in basal-like breast cancer cell lines . Research has shown that CLDND1 knockdown induces caspase-dependent cell death through the intrinsic apoptotic pathway . To investigate this relationship, researchers can implement the following methodological approach:

Experimental Design:

• Establish experimental groups: control cells, CLDND1 siRNA-treated cells, and cells with pharmacological inhibitors of specific apoptotic pathways

• Perform time-course experiments (24h, 48h, 72h post-treatment)

Detection of CLDND1 Expression:

• Use CLDND1 Antibody, HRP conjugated for Western blot analysis to confirm knockdown efficiency

• Quantify expression levels relative to loading controls

Apoptosis Assessment:

• Measure cell viability using WST-1 assay as described in previous studies

• Perform immunoblotting for apoptotic markers (cleaved caspase-3, PARP)

• Assess mitochondrial cytochrome C release by subcellular fractionation followed by immunoblotting

• Evaluate nuclear fragmentation through DAPI staining and fluorescence microscopy

Pathway Analysis:

• Incorporate MEK1/2 inhibitors (U0126) and JNK inhibitors (SP600125) to investigate pathway interactions

• Monitor ERK1/2 phosphorylation status using phospho-specific antibodies alongside CLDND1 detection

• Perform co-immunoprecipitation experiments using CLDND1 Antibody to identify interaction partners

This approach leverages findings that CLDND1 knockdown induces apoptosis that is potentiated by MEK1/2 and JNK inhibition, while ERK1/2 phosphorylation may serve as a compensatory protective mechanism . The HRP-conjugated format allows for direct detection in Western blot and immunoprecipitation applications, simplifying workflow and improving sensitivity in detecting potentially transient protein interactions during apoptosis.

What are the considerations for using CLDND1 Antibody, HRP conjugated in studying cancer biomarkers?

When utilizing CLDND1 Antibody, HRP conjugated for cancer biomarker studies, researchers should consider several methodological aspects based on the complex role of CLDND1 in different cancer types:

Cancer Type-Specific Analysis:

• For lung adenocarcinoma: Focus on CLDND1's role as a cancer invasion/metastasis suppressor

  • Correlate CLDND1 expression with known metastasis suppressors (CTGF, THBS1, DLC1) and enhancers (SPP1, CUTL1, TGF-α)

  • Examine relationship with patient survival data

• For colorectal cancer: Investigate CLDND1's potential role in tumor progression

  • Analyze expression in different stages of colorectal cancer

  • Compare distal vs. proximal colon expression patterns

• For breast cancer: Evaluate CLDND1 as a survival factor

  • Stratify analysis by molecular subtypes (basal-like vs. luminal)

  • Quantify expression levels in relation to cell viability and apoptotic markers

Methodological Approaches:

• Tissue Microarray Analysis:

  • Use CLDND1 Antibody, HRP conjugated at optimized dilutions (1:100-1:200)

  • Score expression levels (0-3+) based on staining intensity and percentage of positive cells

  • Correlate with clinicopathological parameters and patient outcomes

• Circulating Tumor Cell Detection:

  • Develop protocols for CLDND1 detection in liquid biopsies

  • Optimize fixation and permeabilization conditions for circulating cells

• Multiplex Immunohistochemistry:

  • Combine CLDND1 detection with other cancer biomarkers

  • Use spectral unmixing to differentiate multiple chromogenic signals

By tailoring the approach to cancer-specific CLDND1 biology, researchers can maximize the utility of CLDND1 Antibody, HRP conjugated in biomarker discovery and validation studies, potentially identifying new therapeutic targets or prognostic indicators.

How can specificity of CLDND1 Antibody, HRP conjugated be validated in experimental systems?

Rigorous validation of CLDND1 Antibody, HRP conjugated specificity is crucial for generating reliable research data. Implement the following comprehensive validation strategy:

Primary Validation Methods:

• Peptide Competition Assay:

  • Pre-incubate antibody with excess immunizing peptide (5-10-fold molar excess)

  • Process identical samples in parallel (blocked vs. unblocked antibody)

  • Complete disappearance of signal confirms specificity for target epitope

• Genetic Knockdown/Knockout Controls:

  • Utilize siRNA-mediated knockdown of CLDND1 in cell lines (validated siRNAs available from published studies)

  • Compare signal intensity between control and CLDND1-depleted samples

  • Quantify reduction in signal proportional to knockdown efficiency

• Recombinant Protein Expression:

  • Overexpress tagged CLDND1 in a low-expressing cell line

  • Verify signal enhancement corresponding to overexpression level

  • Confirm band shift with tagged constructs

Secondary Validation Methods:

• Multiple Antibody Verification:

  • Compare results with alternative CLDND1 antibodies targeting different epitopes

  • Consistent detection pattern increases confidence in specificity

• Tissue Expression Profiling:

  • Examine CLDND1 expression across tissues known to have differential expression

  • Confirm highest expression in CNS tissues (corpus callosum, cerebral cortex, etc.)

  • Verify lower expression in heart tissue

• Cross-Species Reactivity Testing:

  • Test antibody performance with human, mouse, and rat samples

  • Expected high reactivity due to conserved sequences (mouse - 91%, rat - 89% identity)

Recommended Validation Workflow:

• Initial Western blot analysis with positive and negative control tissues

• Comparison of observed molecular weight (29 kDa) with theoretical prediction

• Peptide competition assay for epitope specificity

• siRNA knockdown verification for target specificity

• Immunohistochemistry pattern analysis in tissues with known expression profiles

Proper validation not only confirms antibody specificity but also establishes optimal working conditions for different experimental applications, ensuring meaningful and reproducible research outcomes.

What are common troubleshooting issues with CLDND1 Antibody, HRP conjugated and their solutions?

When working with CLDND1 Antibody, HRP conjugated, researchers may encounter several technical challenges. The following table presents common issues, their potential causes, and methodological solutions:

Advanced Troubleshooting Approaches:

• For highly sensitive detection needs, consider using amplification systems such as tyramide signal amplification (TSA) compatible with HRP conjugates

• For tissues with high lipid content, incorporate additional permeabilization steps with optimized detergent concentrations

• For multiplex applications, carefully test for potential cross-reactivity with other detection systems

• For quantitative applications, establish standard curves using recombinant CLDND1 protein at known concentrations

How can CLDND1 Antibody, HRP conjugated be utilized in therapeutic target validation studies?

CLDND1 has emerged as a potential therapeutic target in various pathological conditions, particularly in cancer and infectious diseases. CLDND1 Antibody, HRP conjugated can play a pivotal role in therapeutic target validation through the following methodological approaches:

Target Expression and Accessibility Profiling:

• Comprehensive tissue microarray screening to map CLDND1 expression across normal and pathological specimens

• Quantification of membrane-localized versus intracellular CLDND1 pools

• Co-localization studies with tight junction markers to assess accessibility

Functional Validation Approaches:

• Antibody-mediated targeting studies:

  • Compare effects of unconjugated antibodies vs. HRP-conjugated antibodies on cell viability

  • Assess internalization kinetics through time-course immunofluorescence studies

  • Evaluate potential for antibody-dependent cellular cytotoxicity against CLDND1-expressing cells

• Small molecule permeability studies:

  • Leverage CLDND1's role in tight junctions and paracellular permeability

  • Evaluate effects of CLDND1 modulation on drug absorption and distribution

  • Quantify barrier function changes in response to targeted interventions

Cancer-Specific Applications:

• For breast cancer:

  • Compare therapeutic sensitivity in cell lines with varying CLDND1 expression levels

  • Combine CLDND1 targeting with MEK1/2 inhibitors to potentiate apoptosis

  • Develop stratification criteria for potential CLDND1-targeted therapies

• For hepatitis C virus (HCV) infection:

  • Evaluate CLDND1 targeting as an antiviral strategy based on findings with anti-CLDND1 monoclonal antibodies

  • Develop screening assays for compounds that modulate CLDND1 function

Emerging Therapeutic Approaches:

• Near-infrared fluorophore conjugation for fluorescence-guided surgery of colorectal tumors

• Chimeric antibody development based on human-mouse chimeric CLDND1 mAb (clone 3A2)

• Clostridium perfringens enterotoxin (cCPE) variants that bind claudins for targeted delivery

This methodological framework leverages CLDND1 Antibody, HRP conjugated as both a research tool for target validation and as a potential scaffold for therapeutic development, aligning with recent findings on CLDND1's multifaceted roles in disease processes.

What are the considerations for using CLDND1 Antibody, HRP conjugated in multiplex immunoassays?

Implementing CLDND1 Antibody, HRP conjugated in multiplex immunoassay systems requires careful methodological considerations to ensure compatibility, specificity, and optimal signal detection. The following framework addresses key technical aspects:

Platform Selection and Compatibility:

• Chromogenic Multiplex IHC:

  • Select complementary chromogens with spectral separation from DAB (used with HRP)

  • Consider sequential detection protocols with intermediate stripping steps

  • Implement multispectral imaging for signal deconvolution

• Fluorescent Multiplex Systems:

  • Convert HRP signal to fluorescence using tyramide signal amplification (TSA)

  • Select fluorophores with minimal spectral overlap

  • Include autofluorescence quenching steps for tissues with high background

• Protein Array Platforms:

  • Optimize spotting concentration and blocking conditions

  • Validate signal linearity across relevant concentration ranges

  • Include internal calibration standards

Antibody Panel Design:

• Target Selection Considerations:

  • Pair CLDND1 with biologically relevant markers:

    • Tight junction proteins (occludin, ZO-1) for barrier function studies

    • Apoptosis markers (cleaved caspase-3, PARP) for cell death pathway analysis

    • Cancer metastasis regulators (CTGF, THBS1, DLC1, SPP1) for invasion studies

• Technical Compatibility Assessment:

  • Verify antibody species compatibility to prevent cross-reactivity

  • Test for potential antigen masking in sequential staining protocols

  • Establish optimal antibody dilutions in multiplex context (typically higher than single-plex)

Signal Optimization Strategies:

• Signal Amplification:

  • Implement catalyzed reporter deposition (CARD) for low-abundance targets

  • Use polymer detection systems compatible with HRP conjugates

  • Optimize signal development timing to balance sensitivity and specificity

• Background Reduction:

  • Incorporate dual blocking strategies (protein and avidin/biotin if relevant)

  • Include detergent optimization in wash buffers

  • Implement spectral unmixing algorithms for autofluorescence removal

Validation and Quality Control:

• Single-plex vs. Multiplex Comparison:

  • Verify consistent staining patterns between single antibody and panel approaches

  • Quantify potential signal enhancement or suppression in multiplex format

• Cross-platform Validation:

  • Confirm key findings using orthogonal detection methods

  • Correlate multiplex IHC results with quantitative techniques (qPCR, Western blot)

By addressing these methodological considerations, researchers can effectively incorporate CLDND1 Antibody, HRP conjugated into multiplexed assay systems, enabling comprehensive analysis of CLDND1 in complex biological contexts while maintaining specificity and sensitivity.

What future research directions are emerging for CLDND1 antibodies in biomedical research?

The expanding understanding of CLDND1 biology is opening new avenues for research applications of CLDND1 antibodies, including HRP conjugates. Several promising future directions include:

• Single-cell protein profiling: Development of highly sensitive detection systems using CLDND1 antibodies for characterizing heterogeneous cell populations in complex tissues, particularly in CNS research where CLDND1 shows differential expression patterns .

• Therapeutic target validation: Following the emerging evidence of CLDND1's role in cancer biology , antibody-based approaches will be crucial for validating its potential as a druggable target, particularly for personalized medicine applications in breast and lung cancers.

• Pathway interaction mapping: Given CLDND1's involvement in survival pathways and its interaction with MEK/ERK signaling in cancer cells , antibody-based proximity ligation assays could reveal novel protein interaction networks governing cell fate decisions.

• Barrier function regulation: As a claudin family member, CLDND1's role in tissue-specific barrier functions remains to be fully characterized, with potential implications for drug delivery across biological barriers, particularly in the CNS .

• Biomarker validation: The differential expression of CLDND1 in various cancers suggests its potential as a diagnostic or prognostic biomarker, requiring standardized antibody-based detection systems for clinical application development .