CLUAP1 Antibody

What is CLUAP1 and why is it significant in research?

CLUAP1 (Clusterin Associated Protein 1) was originally identified as a protein that interacts with CLU/clusterin, with its gene frequently upregulated in colon cancer . The protein has garnered significant research interest due to its overexpression in multiple cancer types, including osteosarcoma, ovarian, colon, and lung cancers . CLUAP1 is most abundantly expressed in testis among normal tissues, making its differential expression in cancerous states particularly notable . The protein has a calculated molecular weight of 48 kDa but is commonly observed at 53 kDa in experimental conditions, suggesting post-translational modifications . Due to its association with multiple cancer types, CLUAP1 represents a potential prognostic/diagnostic marker and immunotherapy target, particularly for osteosarcoma .

What types of CLUAP1 antibodies are available for research applications?

Two major types of CLUAP1 antibodies are available for research:

• Polyclonal antibodies: Antibodies such as 17470-1-AP target CLUAP1 in multiple applications including Western Blot, Immunofluorescence/ICC, Immunoprecipitation, and ELISA . These antibodies typically show reactivity with human, mouse, rat, and canine samples .

• Monoclonal antibodies: Available as matched antibody pairs (such as catalog MP51001-1) designed specifically for immunoassays . These include:

  • Capture antibody: Clone 1A9G4 (catalog 68353-2-PBS)

  • Detection antibody: Clone 2C11F7 (catalog 68353-3-PBS)

Both formats have specific applications, with polyclonal antibodies offering broader epitope recognition and monoclonal antibodies providing greater specificity.

What are the validated applications for CLUAP1 antibodies?

CLUAP1 antibodies have been validated for multiple research applications:

The application should be selected based on your specific research question, with Western blot being particularly useful for detecting CLUAP1 overexpression in cancer tissue samples.

How can CLUAP1 antibodies be utilized in cancer research studies?

CLUAP1 antibodies serve multiple advanced functions in cancer research:

• Expression profiling: CLUAP1 is overexpressed in several cancer types including osteosarcoma, ovarian, colon, and lung cancers . Researchers can utilize antibodies to quantify this overexpression via Western blot or immunohistochemistry.

• Diagnostic/prognostic marker development: The differential expression of CLUAP1 between normal and cancerous tissues makes it a candidate biomarker, particularly in osteosarcoma . Antibody-based assays can help establish cutoff values for clinical significance.

• Therapeutic target validation: As suggested by studies, CLUAP1 may represent a target for immunotherapy of osteosarcoma . Antibodies can help validate this potential by studying protein interactions and functional effects of CLUAP1 inhibition.

• Cancer tissue characterization: CLUAP1 antibodies in multiplexed immunohistochemistry can help characterize tumor microenvironments and heterogeneity.

These applications contribute to both basic cancer biology understanding and translational research aimed at developing new diagnostic and therapeutic approaches.

What considerations should be made when designing CLUAP1 knockdown/knockout validation experiments?

When designing knockdown/knockout experiments to validate CLUAP1 antibodies:

• Antibody selection: Use antibodies that have been validated in KD/KO experiments previously, as indicated in product literature . This ensures the antibody can detect changes in protein expression.

• Control selection: Include appropriate positive controls (tissues with known high expression like testis) and negative controls (knockdown/knockout samples) .

• Detection method optimization: CLUAP1 is observed at 53 kDa despite a calculated molecular weight of 48 kDa . Ensure your detection system can accurately capture this molecular weight range.

• Multiple validation approaches: Combine protein detection (Western blot) with functional assays to confirm knockdown effects, as CLUAP1 has been implicated in cancer cell biology .

• Cross-reactivity assessment: Verify specificity using samples from different species if working with non-human models, as reactivity has been confirmed in human, mouse, rat, and canine samples .

How can CLUAP1 antibody pairs be employed in developing quantitative immunoassays?

Development of quantitative immunoassays for CLUAP1 requires:

• Matched antibody pair selection: Utilize validated pairs such as the monoclonal matched antibody pair MP51001-1, comprising capture antibody (Clone 1A9G4) and detection antibody (Clone 2C11F7) .

• Assay platform selection:

  • Cytometric bead arrays have been validated with a detection range of 0.391-50 ng/mL

  • ELISA development requires optimization of coating, blocking, and detection conditions

  • Other potential platforms include chemiluminescent immunoassays or multiplex arrays

• Standard curve establishment: Create recombinant CLUAP1 protein standards to generate accurate quantification curves.

• Sample type validation: Validate the assay using relevant biological samples (cancer cell lines, tissue lysates) with known CLUAP1 expression levels.

• Sensitivity and specificity assessment: Determine lower limits of detection and potential cross-reactivity with structurally similar proteins.

These immunoassays can provide valuable quantitative data on CLUAP1 expression in various cancer types, potentially supporting diagnostic applications.

What are the optimal sample preparation techniques for CLUAP1 detection in different applications?

Sample preparation varies by application:

• Western Blot:

  • Use RIPA or NP-40 buffer for cell lysis

  • Include protease inhibitors to prevent degradation

  • Heat samples at 95°C for 5 minutes in reducing sample buffer

  • Load 20-50 μg of total protein per lane

  • Validated samples include mouse testis tissue, COLO 320 cells, HepG2 cells, and Y79 cells

• Immunoprecipitation:

  • Use 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

  • Pre-clear lysates with protein A/G beads

  • Incubate antibody with lysate overnight at 4°C

  • HepG2 cells have been validated for CLUAP1 IP

• Immunofluorescence/ICC:

  • Fix cells with 4% paraformaldehyde (10 minutes, room temperature)

  • Permeabilize with 0.1% Triton X-100

  • Block with 1-5% BSA

  • Use antibody at 1:50-1:500 dilution

  • MDCK cells have been validated for CLUAP1 IF/ICC

• Immunohistochemistry:

  • Use formalin-fixed, paraffin-embedded sections

  • Perform antigen retrieval (citrate buffer, pH 6.0)

  • Block endogenous peroxidase and non-specific binding

  • Apply antibody at optimized dilution (typically 1:100-1:500)

Proper sample preparation is critical for obtaining reliable and reproducible results across all applications.

How should researchers address potential cross-reactivity concerns with CLUAP1 antibodies?

To address cross-reactivity concerns:

• Species verification: While CLUAP1 antibodies show reactivity with human, mouse, rat, and canine samples , always verify specificity when working with less common species or specialized tissues.

• Blocking optimization: Use 5% BSA or 5% non-fat dry milk in TBS-T for Western blots to minimize non-specific binding.

• Validation controls:

  • Positive control: Use tissues known to express CLUAP1, such as testis

  • Negative control: Include samples from knockdown/knockout models

  • Peptide competition: Pre-incubate antibody with immunizing peptide to confirm specificity

• Antibody selection: For critical experiments, consider using antibodies that have been verified on protein arrays containing target protein plus non-specific proteins .

• Dilution optimization: Titrate antibody concentrations (starting with manufacturer recommendations) to determine optimal signal-to-noise ratio in your specific experimental system .

Careful attention to these factors will minimize false positive results and ensure accurate data interpretation.

What storage and handling protocols maximize CLUAP1 antibody performance and longevity?

Optimal storage and handling of CLUAP1 antibodies:

• Storage temperature:

  • Store at -20°C for long-term storage

  • Some antibodies require -80°C storage, particularly monoclonal matched pairs

  • Avoid repeated freeze-thaw cycles

• Aliquoting:

  • Divide antibody solution into single-use aliquots upon receipt

  • For antibodies in glycerol (e.g., those in PBS with 50% glycerol ), aliquoting is less critical but still recommended

• Buffer composition:

  • Typical storage buffers include PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

  • Some preparations may contain 0.1% BSA for stability

• Thawing protocol:

  • Thaw antibodies on ice

  • Centrifuge briefly before opening to collect solution

  • Mix gently by flicking or inverting, avoid vortexing

• Shelf-life:

  • Most antibodies remain stable for one year after shipment when stored properly

  • Monitor for signs of degradation (precipitation, loss of activity)

Following these guidelines will ensure optimal antibody performance throughout your research project.

What are common issues when using CLUAP1 antibodies and how can they be resolved?

Common issues and solutions:

• Weak or absent signal in Western blot:

  • Increase antibody concentration (try 1:500 if 1:1000 shows weak signal)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use enhanced detection systems (high-sensitivity ECL)

  • Ensure target is present (CLUAP1 is highly expressed in testis, but variable in other tissues)

  • Verify sample preparation (adequate lysis and protein extraction)

• Multiple bands or unexpected molecular weight:

  • CLUAP1 is observed at 53 kDa despite a calculated weight of 48 kDa

  • Additional bands may represent isoforms, post-translational modifications, or degradation products

  • Use positive control tissues (testis) to confirm correct banding pattern

  • Optimize gel percentage and running conditions

• High background in immunofluorescence:

  • Increase blocking time/concentration

  • Dilute antibody further (try 1:200 if 1:50 shows high background)

  • Include 0.1% Tween-20 in washing steps

  • Ensure proper fixation and permeabilization

• Inconsistent immunoprecipitation results:

  • Adjust antibody amount (0.5-4.0 μg for 1.0-3.0 mg of total protein)

  • Pre-clear lysates thoroughly

  • Increase wash stringency

  • Confirm expression in input sample via Western blot

Systematic troubleshooting based on these guidelines will help resolve most technical issues.

How can researchers validate the specificity of CLUAP1 antibodies in their experimental systems?

Comprehensive validation approaches include:

• Multiple antibody comparison:

  • Test different antibodies targeting distinct epitopes of CLUAP1

  • Compare polyclonal (e.g., 17470-1-AP) and monoclonal antibodies (e.g., clones 1A9G4 and 2C11F7)

  • Consistent results across antibodies strengthen confidence in specificity

• Genetic validation:

  • Use CLUAP1 knockdown/knockout models

  • Compare wildtype and KD/KO samples by Western blot

  • Loss of signal in KD/KO samples confirms specificity

• Mass spectrometry validation:

  • Perform immunoprecipitation followed by mass spectrometry

  • Verify CLUAP1 identity in pulled-down proteins

  • Analyze any additional proteins for potential cross-reactivity

• Expression pattern correlation:

  • Compare antibody staining patterns with known expression profiles

  • CLUAP1 is highly expressed in testis among normal tissues

  • Overexpression in certain cancer types (osteosarcoma, colon, ovarian, lung)

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide

  • Signal abolishment confirms specificity to the target epitope

These validation approaches ensure reliable and reproducible research outcomes.

What quality control measures should be implemented when using CLUAP1 antibodies in multi-center or longitudinal studies?

For robust multi-center or longitudinal studies:

• Antibody lot consistency:

  • Record lot numbers for all experiments

  • Test each new lot against previous lots

  • Purchase larger quantities of a single lot for extended studies

• Standard operating procedures (SOPs):

  • Develop detailed protocols for each application

  • Specify critical parameters (dilutions, incubation times, temperatures)

  • Distribute identical protocols to all participating centers

• Reference standards:

  • Create standard positive controls (cell lysates, tissue extracts)

  • Distribute identical controls to all centers

  • Use recombinant CLUAP1 protein as quantitative standard

• Centralized testing:

  • Consider periodic centralized testing of key samples

  • Implement proficiency testing between centers

  • Analyze inter-laboratory variation

• Data normalization strategy:

  • Define normalization controls (loading controls for Western blot)

  • Establish quantification methods (densitometry parameters)

  • Use consistent image acquisition settings

• Documentation requirements:

  • Maintain detailed records of antibody storage conditions

  • Document any deviations from protocols

  • Record raw data and analysis methods

How might CLUAP1 antibodies contribute to cancer immunotherapy development?

CLUAP1 antibodies could advance cancer immunotherapy in several ways:

• Target validation: CLUAP1 has been identified as a potential immunotherapy target, particularly for osteosarcoma . Antibodies can confirm its accessibility on cancer cell surfaces and validate its differential expression between tumor and normal tissues.

• Epitope mapping: Using different monoclonal antibodies targeting various epitopes can identify the most immunogenic regions of CLUAP1, informing vaccine design or antibody-drug conjugate development.

• Therapeutic antibody development: The validated antibody pairs provide starting points for developing therapeutic antibodies targeting CLUAP1 in cancer cells.

• Biomarker identification: As CLUAP1 is overexpressed in multiple cancer types , antibody-based detection can help identify patients most likely to respond to CLUAP1-targeted therapies.

• Functional studies: Antibodies can help elucidate the role of CLUAP1 in cancer cell biology, potentially revealing additional therapeutic targets in the same pathway.

The connection between CLUAP1 and cancer makes antibodies against this protein valuable tools in advancing immunotherapeutic approaches.

What methodological innovations might improve CLUAP1 detection sensitivity and specificity?

Emerging technologies to enhance CLUAP1 detection:

• Single-cell protein analysis:

  • Mass cytometry (CyTOF) incorporation of CLUAP1 antibodies

  • Single-cell Western blotting for heterogeneity assessment

  • Imaging mass cytometry for spatial context in tissues

• Proximity-based detection methods:

  • Proximity ligation assays (PLA) to study CLUAP1 interactions with clusterin

  • FRET-based assays for real-time interaction studies

  • BiFC (Bimolecular Fluorescence Complementation) for live-cell visualization

• Nanobody development:

  • Creation of CLUAP1-specific nanobodies for improved tissue penetration

  • Smaller binding domains for accessing restricted epitopes

  • Potential for intracellular expression and tracking

• Multiplexed detection platforms:

  • Simultaneous detection of CLUAP1 with other cancer biomarkers

  • Digital spatial profiling for tumor microenvironment analysis

  • Multiplex immunofluorescence for co-localization studies

• Enhanced sensitivity approaches:

  • Signal amplification methods (tyramide signal amplification)

  • Ultra-sensitive ELISA formats (Single Molecule Array technology)

  • Quantum dot-conjugated antibodies for improved signal-to-noise ratios

These methodological innovations could significantly advance our understanding of CLUAP1's role in cancer biology and improve diagnostic capabilities.