Ckap4 Antibody

What is CKAP4 and why is it significant in cancer research?

CKAP4 (cytoskeleton-associated protein 4) is a 63 kDa cell surface receptor protein that functions as a receptor for Dickkopf 1 (DKK1), a secreted protein. The DKK1–CKAP4 signaling pathway has significant implications in oncology as it is activated in various malignant tumors, including pancreatic, lung, esophageal, and liver cancers, where it promotes tumor growth and proliferation. This pathway represents a novel molecular target for cancer therapy, making CKAP4 antibodies increasingly important in translational cancer research. The protein is also known as Climp-63 and has roles in associating triads and microtubules as a partner of triadin.

What are the different types of CKAP4 antibodies available for research?

Several types of CKAP4 antibodies have been developed for research purposes, including:

• Mouse anti-CKAP4 antibodies (mCKAP4 Ab), such as the original 3F11-2B10 and recombinant versions

• Humanized anti-CKAP4 antibody (hCKAP4 Ab), notably the Hv1Lt1 developed from the 3F11-2B10 nucleotide sequence

• Polyclonal antibodies like 16686-1-AP (unconjugated) and CL594-16686 (CoraLite®594 conjugated)

These antibodies vary in their host species, isotype, and conjugation status, offering researchers flexibility for different experimental applications.

What are the optimal conditions for using CKAP4 antibody in Western Blot applications?

For Western Blot applications using CKAP4 antibody (16686-1-AP), the recommended dilution range is 1:5000-1:50000. This antibody has been successfully tested with various sample types including A431 cells, rat kidney tissue, HeLa cells, HepG2 cells, and NIH/3T3 cells. When working with CKAP4 in Western Blot, researchers should expect to observe a band at approximately 63 kDa, which corresponds to the observed molecular weight of CKAP4, though its calculated molecular weight is 66 kDa (602 amino acids). It's advisable to include appropriate positive controls from the validated cell lines and optimize the antibody concentration based on your specific experimental system.

How should CKAP4 antibodies be applied in immunofluorescence studies?

For immunofluorescence (IF) and immunocytochemistry (ICC) applications, CKAP4 antibodies can be used at a dilution range of 1:50-1:500. Both the unconjugated (16686-1-AP) and the CoraLite®594-conjugated (CL594-16686) versions have been validated for IF/ICC applications, particularly in HepG2 cells. When using the fluorescent-conjugated antibody (CL594-16686), it's important to note its excitation/emission maxima wavelengths of 588 nm/604 nm and take precautions to avoid light exposure during storage and handling. For optimal results, specific protocols are available for download from the manufacturer, and researchers should titrate the antibody concentration in their specific experimental systems.

What protocols are recommended for CKAP4 antibody in immunohistochemistry?

For immunohistochemistry (IHC) applications using CKAP4 antibody (16686-1-AP), the recommended dilution range is 1:50-1:500. This antibody has been positively validated in human kidney and liver tissues. For optimal antigen retrieval, TE buffer at pH 9.0 is suggested, though citrate buffer at pH 6.0 can be used as an alternative. The antibody is suitable for formalin-fixed, paraffin-embedded tissue sections. When establishing the IHC protocol, researchers should optimize staining conditions for their specific tissue samples, including fixation method, antigen retrieval conditions, antibody concentration, and incubation times. As with all antibody applications, appropriate positive and negative controls should be included.

How does the humanized anti-CKAP4 antibody (Hv1Lt1) compare to mouse antibodies in pancreatic cancer models?

The humanized anti-CKAP4 antibody (Hv1Lt1) has demonstrated superior binding affinity for CKAP4 compared to the original mouse antibody (3F11-2B10). In functional studies, Hv1Lt1 effectively inhibits DKK1 binding to CKAP4, suppresses AKT activity, and inhibits sphere formation of pancreatic cancer cells at levels comparable to 3F11-2B10. In xenograft tumor models using human pancreatic cancer cells, Hv1Lt1 successfully suppressed tumor formation. Additionally, in murine cancer models where pancreatic cancer organoids were orthotopically transplanted into the pancreas, Hv1Lt1 demonstrated significant inhibition of tumor growth. The humanized antibody also appears to modulate anti-tumor immune reactions, leading to increased infiltration of cytotoxic T cells in the tumor microenvironment, suggesting it may enhance immune-mediated tumor suppression in addition to its direct effects on cancer cells.

What is the mechanism by which anti-CKAP4 antibodies inhibit tumor growth?

Anti-CKAP4 antibodies inhibit tumor growth through multiple mechanisms:

• Direct pathway inhibition: The antibodies block the binding of DKK1 to CKAP4, preventing activation of the DKK1-CKAP4 signaling pathway that normally promotes cancer cell proliferation.

• Suppression of AKT activity: By interrupting the signaling cascade, the antibodies reduce AKT activation, a key driver of cell survival and proliferation in cancer cells.

• Inhibition of cancer stem cell properties: The antibodies inhibit sphere formation, which is a measure of cancer stem cell self-renewal and proliferative capacity.

• Immune modulation: In tumor samples from mice treated with Hv1Lt1, researchers observed modulation of anti-tumor immune reactions, with increased infiltration of cytotoxic T cells in the tumor microenvironment, suggesting the antibody may also enhance immune-mediated tumor suppression.

• Synergistic effects: When combined with other chemotherapy drugs, anti-CKAP4 antibodies have shown stronger anti-tumor effects compared to monotherapy, indicating potential for combination therapeutic approaches.

What are the potential advantages of combining anti-CKAP4 antibody therapy with conventional chemotherapeutics?

Combining anti-CKAP4 antibody therapy with conventional chemotherapeutics offers several potential advantages:

• Enhanced efficacy: Research has demonstrated that combination of Hv1Lt1 with other chemotherapy drugs exhibits stronger anti-tumor effects compared to monotherapy, likely due to targeting of multiple cancer survival and proliferation pathways simultaneously.

• Complementary mechanisms of action: While conventional chemotherapeutics typically target rapidly dividing cells through various mechanisms (DNA damage, mitotic inhibition, etc.), anti-CKAP4 antibodies specifically inhibit the DKK1-CKAP4 signaling pathway. This complementary approach may address multiple aspects of tumor biology.

• Potential to overcome resistance: Cancer cells often develop resistance to single-agent therapies. Combination approaches that target different pathways may reduce the likelihood of resistance development.

• Immune system engagement: The observed increase in cytotoxic T cell infiltration in tumors treated with anti-CKAP4 antibodies suggests these antibodies may enhance immune surveillance, potentially augmenting the effects of immunomodulatory chemotherapies.

• Possibility for dose reduction: Effective combinations might allow for reduced doses of conventional chemotherapeutics, potentially decreasing toxic side effects while maintaining therapeutic efficacy.

How can researchers address inconsistent results when using CKAP4 antibodies in different cell lines?

When encountering inconsistent results with CKAP4 antibodies across different cell lines, researchers should consider several factors:

• Expression levels: CKAP4 expression varies across cell types. Verify CKAP4 expression levels in your cell lines using RT-qPCR or published databases before antibody applications.

• Antibody validation: Ensure the antibody has been validated for your specific cell type. The search results indicate successful detection in specific cell lines (A431, HeLa, HepG2, NIH/3T3) and tissues (rat kidney, human kidney, human liver).

• Optimization of protocols: Different cell lines may require adjusted protocols:

  • Lysate preparation: Optimize lysis buffer composition for your cell type

  • Fixation conditions: For IF/IHC applications, test different fixatives and durations

  • Antigen retrieval: For IHC, compare TE buffer (pH 9.0) versus citrate buffer (pH 6.0)

• Antibody titration: The manufacturer recommends titrating the antibody in each testing system. Try a dilution series within the recommended range (e.g., 1:50-1:500 for IF/ICC)

• Post-translational modifications: Consider that CKAP4 might undergo different post-translational modifications in different cell types, potentially affecting antibody recognition.

What controls should be included when validating CKAP4 antibody specificity in a new experimental system?

When validating CKAP4 antibody specificity in a new experimental system, include these essential controls:

• Positive controls:

  • Known CKAP4-expressing cell lines: Use validated cell lines like HeLa, HepG2, A431, or NIH/3T3 cells

  • Recombinant CKAP4 protein: If available, use as a positive control in Western blot

• Negative controls:

  • Isotype control: Include a matched isotype antibody (rabbit IgG for most CKAP4 antibodies) to assess non-specific binding

  • CKAP4 knockdown/knockout: Use CKAP4 siRNA, shRNA, or CRISPR-modified cells as specificity controls

  • Secondary antibody only: Omit primary antibody to assess secondary antibody background

• Cross-reactivity assessment:

  • Test multiple CKAP4 antibodies targeting different epitopes if possible

  • Confirm the expected molecular weight (approximately 63 kDa)

• Blocking peptide controls:

  • When available, pre-incubate the antibody with the immunizing peptide before application to demonstrate specificity

• Application-specific controls:

  • For IF/ICC: Include counterstains to confirm expected subcellular localization

  • For IP: Include non-immunoprecipitated lysate (input) as comparison

  • For IHC: Include tissues with known CKAP4 expression patterns

How should researchers interpret differences between calculated (66 kDa) and observed (63 kDa) molecular weights of CKAP4?

The discrepancy between the calculated molecular weight of CKAP4 (66 kDa) and its observed molecular weight (63 kDa) on Western blots is not uncommon in protein analysis and can be attributed to several factors:

• Post-translational modifications:

  • Proteolytic processing: CKAP4 may undergo specific cleavage events that remove small portions of the protein

  • Other modifications: Phosphorylation, glycosylation, or other modifications can influence protein migration

• Protein structure:

  • Compact folding: Proteins with compact tertiary structures may migrate faster than predicted

  • Hydrophobicity: Highly hydrophobic proteins often bind more SDS and migrate faster than expected

• Technical considerations:

  • Gel percentage and buffer system: Different electrophoresis conditions can affect protein migration

  • Molecular weight markers: Calibration issues with molecular weight standards may influence apparent size

  • Gel running conditions: Temperature, voltage, and duration can affect migration patterns

• Isoform expression:

  • Alternative splicing: Cell types may express different CKAP4 isoforms with varying molecular weights

  • Tissue-specific modifications: Different tissues may process CKAP4 differently

When interpreting results, researchers should consider these factors and focus on consistent detection at the 63 kDa range rather than expecting precise alignment with the calculated molecular weight. Additional validation techniques such as mass spectrometry can help confirm protein identity when questions arise.

What are promising research avenues for expanding the therapeutic applications of anti-CKAP4 antibodies beyond pancreatic cancer?

Given the role of the DKK1-CKAP4 pathway in multiple cancer types, several promising research avenues exist for expanding anti-CKAP4 antibody applications:

• Additional cancer indications: Since DKK1-CKAP4 signaling is activated in lung, esophageal, and liver cancers, investigating anti-CKAP4 antibodies in these malignancies represents a logical extension of current research.

• Combination therapies: Further exploration of synergistic combinations with:

  • Immune checkpoint inhibitors: Given the observed immune modulation effects

  • Targeted therapies: Combining with drugs targeting complementary pathways

  • Conventional chemotherapeutics: Expanding on the demonstrated enhanced effects

• Antibody engineering:

  • Bispecific antibodies targeting both CKAP4 and other cancer-associated antigens

  • Antibody-drug conjugates (ADCs) to deliver cytotoxic payloads specifically to CKAP4-expressing cells

  • Enhanced effector functions through Fc engineering to amplify immune responses

• Biomarker development:

  • Identifying patient subpopulations most likely to respond to anti-CKAP4 therapy

  • Developing companion diagnostics to measure DKK1 and CKAP4 expression levels

• Resistance mechanisms:

  • Investigating potential resistance mechanisms to anti-CKAP4 therapy

  • Developing strategies to overcome or prevent resistance

• Understanding the broader role of CKAP4:

  • Investigating CKAP4's functions beyond the DKK1 pathway

  • Exploring the relationship between CKAP4's roles in the cytoskeleton and cancer progression

What methodological approaches could improve the efficacy of anti-CKAP4 antibodies in preclinical and clinical settings?

Several methodological approaches could enhance anti-CKAP4 antibody efficacy:

• Improved antibody delivery systems:

  • Nanoparticle encapsulation to enhance tumor penetration

  • Tumor-targeting strategies to increase local concentration

  • Blood-brain barrier crossing technologies for potential central nervous system applications

• Advanced preclinical models:

  • Patient-derived xenografts that better reflect tumor heterogeneity

  • Humanized mouse models with intact immune systems

  • 3D organoid cultures for high-throughput screening

  • In silico modeling to predict optimal dosing and combinations

• Mechanistic enhancements:

  • Structure-based optimization of antibody binding domains

  • Engineering for increased half-life and tissue penetration

  • Enhancing antibody-dependent cellular cytotoxicity (ADCC) properties

• Biomarker-guided approaches:

  • Identifying predictive biomarkers of response

  • Real-time monitoring of target engagement

  • Development of imaging techniques to track antibody distribution

• Combination strategy refinement:

  • Sequence-specific combinations (concurrent vs. sequential therapy)

  • Dose-finding studies to minimize toxicity while maximizing efficacy

  • Rational combinations based on mechanistic synergy rather than empirical testing

• Translation to clinical settings:

  • Phase 0 microdosing studies to assess pharmacokinetics early

  • Adaptive trial designs to more rapidly identify effective approaches

  • Basket trials enrolling patients based on CKAP4/DKK1 expression rather than cancer type

How might single-cell analysis technologies advance our understanding of CKAP4 function in the tumor microenvironment?

Single-cell analysis technologies offer unprecedented resolution to understand CKAP4's role in cancer:

• Heterogeneity mapping:

  • Single-cell RNA sequencing to identify subpopulations of tumor cells with varying CKAP4 expression

  • Correlation of CKAP4 expression with cancer stem cell markers and drug resistance signatures

  • Identification of rare cell populations that might drive relapse after therapy

• Spatial context understanding:

  • Spatial transcriptomics to map CKAP4 and DKK1 expression within the tumor architecture

  • Multiplexed immunofluorescence to visualize CKAP4 expression relative to immune cells, stromal cells, and vascular structures

  • Analysis of CKAP4 expression at the tumor invasion front versus tumor core

• Dynamic signaling analysis:

  • Single-cell phosphoproteomics to trace DKK1-CKAP4 signaling cascades at individual cell resolution

  • Live-cell imaging with fluorescent reporters to monitor pathway activation in real-time

  • Mass cytometry (CyTOF) to simultaneously measure multiple signaling nodes along with CKAP4 expression

• Immune interaction characterization:

  • Single-cell analysis of tumor-infiltrating lymphocytes in relation to CKAP4-expressing cells

  • T-cell receptor sequencing to identify clonal expansion in response to anti-CKAP4 therapy

  • Analysis of immune checkpoint expression in relation to CKAP4/DKK1 pathway activity

• Therapeutic response prediction:

  • Ex vivo drug sensitivity testing on single cells with varying CKAP4 expression

  • Trajectory analysis to understand cellular state transitions during treatment

  • Identification of resistance-associated cell states before clinical resistance emerges

• Developmental insights:

  • Understanding the normal developmental role of CKAP4 through lineage tracing

  • Comparison of CKAP4 function in normal stem cells versus cancer stem cells

These single-cell approaches would significantly advance our understanding of how the DKK1-CKAP4 pathway operates within the complex tumor ecosystem and potentially reveal new therapeutic strategies.