CLT2 Antibody

What are the validated applications for CLTA antibody in protein detection?

CLTA antibody (10852-1-AP) has been validated for multiple experimental applications with specific reactivity against human, mouse, and rat samples. According to validation data, this antibody can be reliably used for Western blot (WB), immunoprecipitation (IP), immunohistochemistry (IHC), and immunofluorescence/immunocytochemistry (IF/ICC) applications . The antibody has demonstrated positive Western blot detection in multiple cell lines including HEK-293, K-562, Raji, C2C12, C6, and PC-12 cells, as well as mouse brain tissue . For immunoprecipitation, it has shown positive results specifically with mouse brain tissue, making it valuable for neurological research applications .

What dilution ranges should be used for different experimental applications?

The optimal antibody dilution varies significantly depending on the specific application:

It is important to note that these ranges provide starting points, and researchers should optimize the dilution for their specific experimental system and samples . Application-specific optimization is particularly important as sensitivity can vary between tissue types and experimental conditions.

What are the proper storage conditions for maintaining CLTA antibody activity?

The CLTA antibody should be stored at -20°C in PBS buffer containing 0.02% sodium azide and 50% glycerol (pH 7.3) . When properly stored, the antibody remains stable for at least one year after shipment. For the 20μl size format, the product contains 0.1% BSA as a stabilizer. Importantly, aliquoting is not necessary for -20°C storage, which simplifies laboratory handling procedures . Avoiding repeated freeze-thaw cycles is still recommended as a general best practice to maintain antibody performance.

How should antigen retrieval be performed for immunohistochemistry with CLTA antibody?

For optimal immunohistochemical detection of CLTA in formalin-fixed paraffin-embedded tissues, the recommended antigen retrieval protocol involves using TE buffer at pH 9.0 . As an alternative approach, citrate buffer at pH 6.0 may also be used for antigen retrieval if the primary method does not yield satisfactory results . Similar to protocols used for other immunohistochemical assays, automated immunostaining using systems like Leica BOND-III can enhance standardization and reproducibility, as demonstrated in related antibody studies .

What is the expected molecular weight for CLTA detection in Western blot analysis?

Though CLTA has a calculated molecular weight of 27 kDa based on its amino acid sequence, the observed molecular weight in experimental conditions typically ranges between 35-38 kDa . This discrepancy between calculated and observed molecular weights is important to note when interpreting Western blot results, as it likely reflects post-translational modifications such as phosphorylation or glycosylation of the native protein. Researchers should use appropriate molecular weight markers and positive controls to confirm band identity.

How can I validate the specificity of CLTA antibody in my experimental system?

Validating antibody specificity is critical for ensuring reliable experimental results. For CLTA antibody, a multi-faceted validation approach is recommended:

• Knockout/knockdown controls: The antibody has been cited in at least one publication utilizing knockdown/knockout methodology . Testing the antibody in CLTA-knockout or CLTA-depleted samples via siRNA/shRNA should show diminished or absent signal.

• Cross-reactivity assessment: Although the antibody shows reactivity with human, mouse, and rat samples, testing specificity against related proteins (such as CLTB, clathrin heavy chain) is recommended to confirm absence of cross-reactivity.

• Multiple detection techniques: Verify protein detection using complementary methods (e.g., WB, IHC, IF) to confirm consistent target recognition across experimental platforms.

• Immunoprecipitation followed by mass spectrometry: For absolute confirmation of specificity, perform IP with the CLTA antibody followed by mass spectrometric analysis of the pulled-down proteins.

Similar validation approaches have been successfully applied for other antibodies in research contexts, as demonstrated by the methodologies described for CLDN18.2 immunohistochemistry assays .

What are the critical parameters for quantitative flow cytometry using antibodies against clathrin-related proteins?

While the search results don't provide CLTA-specific flow cytometry protocols, principles from related antibody-based flow cytometry can be applied. Based on standardized flow cytometry techniques described in related research:

• Sample preparation: Optimal cell concentration should be approximately 3.0 × 10^5 cells per sample for staining .

• Staining conditions: Incubate cells with antibodies in the dark for 15 minutes at 4°C, followed by two wash steps with PBS containing 1.0% FBS .

• Multiparameter analysis: When investigating clathrin-mediated endocytosis, consider co-staining with related endocytic markers such as AP-2 or dynamin to provide contextual information.

• Data analysis: Use contemporary flow cytometry software such as FlowJo (version 10.5 or later) for comprehensive analysis of results .

• Controls: Include both isotype controls and biological controls (cells known to express high or low levels of CLTA) to establish gating strategies and confirm specificity.

How do monoclonal and polyclonal CLTA antibodies compare in terms of experimental applications and sensitivity?

The CLTA antibody (10852-1-AP) is a polyclonal antibody raised in rabbit . When selecting between polyclonal and monoclonal antibodies for CLTA detection, several research considerations emerge:

Polyclonal Advantages:

• Recognition of multiple epitopes provides robust signal amplification

• Often more tolerant to minor protein denaturation

• Typically effective across multiple applications (WB, IP, IHC, IF)

Monoclonal Considerations:

• Higher consistency between production batches

• May offer superior specificity for distinguishing between closely related proteins

• Often preferred for quantitative applications requiring precise epitope targeting

For quantitative studies, research indicates that monoclonal antibodies with known binding affinities provide more consistent results. For instance, analysis of monoclonal antibodies has shown that binding constants can vary significantly between antibody formats (e.g., IgG vs. Fab fragments), directly impacting experimental outcomes . The search results indicate that the association constant of IgG2a format (4.7 × 10^9 M^-1) was approximately 30-fold higher than Fab fragments in certain experimental contexts .

What approaches can resolve contradictory results when using CLTA antibody across different experimental systems?

When faced with contradictory results across experimental systems, a systematic troubleshooting approach should include:

• Epitope accessibility assessment: The conformation of CLTA may differ between applications. For instance, formaldehyde fixation has been shown to significantly reduce epitope recognition in some antibody systems . Consider alternative fixation methods or antigen retrieval approaches.

• Signal amplification strategies: For low-expression samples, explore signal enhancement through techniques such as tyramide signal amplification for IHC or IF applications.

• Expression level verification: Utilize qPCR to confirm CLTA mRNA expression levels in the experimental system, which can help determine if contradictory protein detection results reflect true biological variation or technical limitations.

• Cross-validation with multiple antibodies: Testing multiple antibodies targeting different epitopes of CLTA can help confirm if contradictory results reflect epitope masking or true protein expression differences.

• Post-translational modification analysis: Consider that post-translational modifications may affect antibody binding, particularly when comparing results across different cell types or experimental conditions.

Research has demonstrated that even well-characterized antibodies can exhibit variable results depending on sample preparation methods. For example, studies comparing untreated cells versus formaldehyde-fixed cells have shown dramatically different antibody binding efficiencies and activation capacities .

How can I optimize CLTA antibody for co-immunoprecipitation experiments investigating clathrin-associated protein complexes?

Optimizing co-immunoprecipitation (co-IP) protocols for CLTA requires careful consideration of several parameters:

• Lysis buffer composition: Use buffers that preserve protein-protein interactions while effectively extracting membrane-associated clathrin complexes. Consider buffers containing:

  • 50 mM Tris-HCl (pH 7.4)

  • 150 mM NaCl

  • 1% NP-40 or IGEPAL CA-630

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

• Antibody amount optimization: For CLTA antibody, use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate . This range provides a starting point, but titration experiments should be performed to identify the optimal ratio for your specific experimental system.

• Pre-clearing step: To reduce non-specific binding, pre-clear lysates with protein A/G beads before adding the CLTA antibody.

• Incubation conditions: For capturing transient interactions, consider shorter incubation times (2-4 hours) at 4°C; for more stable complexes, overnight incubation may yield better results.

• Washing stringency: Balance between removing non-specific interactions and preserving specific ones by testing different wash buffer compositions and washing frequencies.

• Elution strategy: For downstream applications like mass spectrometry, consider native elution using competing peptides rather than denaturing elution with SDS sample buffer.

The IP procedure has been successfully validated using mouse brain tissue , suggesting that neural tissues provide a reliable source of CLTA protein for establishing and optimizing co-IP protocols.

What are the key considerations when designing experiments to study CLTA in different cellular contexts?

When designing experiments to investigate CLTA across different cellular contexts, researchers should consider:

• Endogenous expression levels: Preliminary Western blot analysis should be performed to determine baseline CLTA expression across candidate cell lines. Published validation shows detectable CLTA expression in HEK-293, K-562, Raji, C2C12, C6, and PC-12 cells .

• Subcellular localization patterns: CLTA distribution may vary between cell types. Immunofluorescence studies should be designed to capture potential differences in distribution patterns, with HepG2 and HEK-293 cells serving as validated positive controls for IF applications .

• Functional redundancy: Consider potential compensation by CLTB (clathrin light chain B) when designing knockout or knockdown experiments, as functional redundancy between clathrin light chains may mask phenotypes in single knockout models.

• Tissue-specific isoforms: Account for potential tissue-specific isoforms or post-translational modifications that might affect antibody recognition in experimental designs spanning multiple tissue types.

• Stress conditions: Include experimental conditions that modulate clathrin-mediated endocytosis (e.g., hyperosmotic shock, potassium depletion) to assess functional impacts on CLTA dynamics.

How should I analyze contradictory antibody test results between different experimental techniques?

When faced with contradictory results between techniques (e.g., positive Western blot but negative IHC), a structured analytical approach is essential:

• Epitope conformation analysis: Consider that the CLTA epitope recognized by the antibody may be differentially accessible in various experimental conditions. For example, the immunogen used to create this antibody was a CLTA fusion protein (Ag1299) , which may present epitopes differently than native protein in fixed tissues.

• Cross-validation with genomic approaches: Complement protein-level analysis with RNA-level detection methods such as RT-PCR or RNA-seq to verify gene expression independently of antibody-based detection.

• Method-specific controls: Include application-specific positive and negative controls for each technique. For example, when validating IHC results, human breast cancer tissue has been verified as a positive control for CLTA antibody .

• Quantitative comparison: When possible, perform quantitative analysis (e.g., densitometry for WB, fluorescence intensity measurements for IF) to determine if discrepancies reflect differences in detection sensitivity rather than true presence/absence of the target.

• Literature comparison: Compare results with published literature using systematic review approaches similar to those employed for evaluating clinical efficacy of antibody-based therapies .

What methodological approaches can distinguish between specific antibody binding and background signals?

Distinguishing specific signals from background requires rigorous controls and analytical approaches:

• Blocking optimization: Titrate blocking reagents (BSA, normal serum, commercial blocking buffers) to determine optimal concentrations that minimize background while preserving specific signal.

• Absorption controls: Pre-incubate CLTA antibody with recombinant CLTA protein before application to the sample; specific signals should be abolished while non-specific background persists.

• Isotype controls: Use matched isotype controls (rabbit IgG for CLTA antibody 10852-1-AP) at the same concentration to assess non-specific binding contributed by the antibody class itself .

• Signal-to-noise ratio analysis: Quantify signal intensity in known positive areas compared to known negative areas to establish a reliable threshold for specific detection.

• Secondary antibody-only controls: Perform control staining with secondary antibody alone to identify potential direct binding of detection reagents to the sample.

• Fluorophore spectrum analysis: For fluorescence applications, analyze spectral characteristics of observed signals to distinguish specific fluorescence from autofluorescence, particularly important in tissues with high endogenous fluorescence like neural tissue.

How can I integrate CLTA antibody-based assays with functional studies of clathrin-mediated endocytosis?

Integrating CLTA antibody detection with functional endocytosis assays provides more comprehensive insights:

• Live-cell imaging: Correlate fixed-cell CLTA antibody staining patterns with live-cell dynamics using fluorescently-tagged cargo proteins known to undergo clathrin-mediated endocytosis.

• Endocytic cargo uptake assays: Combine CLTA immunostaining with quantitative measurements of transferrin, EGF, or LDL uptake to correlate CLTA expression levels with functional endocytosis rates.

• Super-resolution microscopy: Employ techniques such as STORM or STED microscopy with CLTA antibody to visualize clathrin-coated pit morphology at nanoscale resolution and correlate with cargo uptake efficiency.

• Pharmacological perturbations: Use endocytosis inhibitors (e.g., dynasore, pitstop-2) in combination with CLTA immunostaining to establish causative relationships between observed structures and functional outcomes.

• Proximity ligation assays: Combine CLTA antibody with antibodies against putative interaction partners to visualize and quantify protein-protein interactions in situ, providing both localization and interaction data simultaneously.

How can CLTA antibody-based techniques be applied to study neurological disorders?

Clathrin plays critical roles in synaptic vesicle recycling and neurotransmitter receptor trafficking, making CLTA antibody valuable for neurological disorder research:

• Brain tissue analysis: The CLTA antibody has been validated in mouse brain tissue for both Western blot and immunoprecipitation applications , making it suitable for examining CLTA expression and interactions in neurological disease models.

• Synaptic function assessment: Combine CLTA immunostaining with markers of synaptic vesicles and active zones to assess potential alterations in synaptic vesicle recycling machinery in disease states.

• Receptor trafficking analysis: Use CLTA antibody in conjunction with antibodies against neurotransmitter receptors to investigate potential dysregulation of receptor endocytosis in conditions such as epilepsy or neurodegenerative disorders.

• Comparative expression studies: Apply validated antibody dilutions (1:5000-1:50000 for WB; 1:50-1:500 for IHC) to compare CLTA expression levels between control and pathological tissues .

• Co-immunoprecipitation studies: Leverage the validated IP application of CLTA antibody to identify altered protein interactions in neurological disease models, using 0.5-4.0 μg of antibody per 1.0-3.0 mg of brain tissue lysate .

What methodological considerations are important when using CLTA antibody in cancer research applications?

When applying CLTA antibody in cancer research, several methodological considerations should be addressed:

• Tissue microarray validation: For high-throughput screening of CLTA expression across multiple tumor samples, validate antibody performance on tissue microarrays containing both normal and malignant tissues.

• Correlation with endocytic activity: As clathrin-mediated endocytosis regulates receptor signaling relevant to cancer (e.g., EGFR, HER2), design experiments that correlate CLTA expression with receptor trafficking dynamics in tumor cells.

• Patient-derived xenograft models: Validate antibody performance in patient-derived xenograft tissues to ensure reliable detection in these increasingly important cancer research models.

• Comparison with prognostic markers: Design multiparameter IHC studies that combine CLTA detection with established prognostic markers to investigate potential correlations.

• Human tissue optimization: The CLTA antibody has been validated in human breast cancer tissue for IHC applications , providing a starting point for optimization in oncology research applications.

How should researchers interpret CLTA antibody signal variations in the context of disease-related studies?

When interpreting CLTA signal variations in disease contexts, researchers should consider: