CLEC3A Antibody

What is the role of CLEC3A in cartilage and bone biology, and which antibody formats are most effective for studying its function?

CLEC3A (C-type lectin domain family 3 member A) is a 22.2 kDa protein that plays crucial roles in cartilage and bone homeostasis. Research shows CLEC3A is highly expressed in cartilage, intervertebral discs, chondrocytes, and nucleus pulposus cells compared to osteoblasts and bone tissue . Its functions include:

• Supporting chondrocyte differentiation

• Maintaining nucleus pulposus cell function

• Enhancing cartilage-specific extracellular matrix architecture

• Accelerating cell proliferation via PI3K-AKT pathway activation

For effective study of CLEC3A:

• Polyclonal antibodies perform well in tissue-based applications (IHC)

• For subcellular localization studies, antibodies targeting the C-terminal region (aa 50 to C-terminus) show high specificity

• Western blot applications typically require 1μg/mL concentration for optimal detection

When studying CLEC3A in cartilage, validation using alcian blue staining as a parallel assay helps confirm antibody specificity by correlating with matrix accumulation .

How do researchers optimize CLEC3A antibody use in immunohistochemistry protocols for different tissue types?

Optimizing CLEC3A antibody protocols requires tissue-specific considerations:

For cartilage tissue:

• Antigen retrieval: Heat citrate buffer (pH 6.0) at 100°C for 20 minutes

• Blocking: 3% hydrogen peroxide in methanol for 30 minutes

• Antibody dilution: 1:5000-1:10000 for rabbit polyclonal antibodies

• Incubation time: 1 hour at room temperature

• Detection method: Streptavidin-biotin-peroxidase method recommended for highest sensitivity

For intervertebral disc tissue:

• Paraffin sections: 4-μm thick

• After deparaffinization, triple wash in PBS is essential

• Apply blocking buffer prior to primary antibody

Evaluation scoring system:

• Four-grade system combining intensity (0-3) and percentage positivity (0-3)

• Positive expression threshold score: ≥3

This scoring method has been validated in studies examining CLEC3A expression in pancreatic neuroendocrine tumors with significantly higher recurrence rates observed in patients with positive CLEC3A expression (P = 0.028) .

What methodological approaches should be employed when using CLEC3A antibodies to investigate the PI3K-AKT signaling pathway?

When investigating CLEC3A's role in PI3K-AKT signaling:

Recommended experimental design:

• Paired overexpression and knockdown experiments:

  • siRNA-mediated CLEC3A knockdown

  • Lentiviral CLEC3A overexpression

• Key targets to measure by western blotting:

  • Total AKT1 protein level (baseline control)

  • Phosphorylated AKT (p-AKT) as activation indicator

  • Downstream targets: PCNA, CCND1, HIF1α

• Pathway verification approach:

  • Apply MK-2206 (PI3K-AKT inhibitor) to CLEC3A-overexpressing cells

  • Monitor reversal of proliferation effects

  • Measure changes in PCNA and CCND1 expression

Typical findings pattern:

This methodology has revealed that while CLEC3A does not change total AKT1 levels, it significantly stimulates AKT phosphorylation, representing the activated state of PI3K-AKT signaling .

How can researchers effectively use CLEC3A antibodies to study autoimmune components in osteoarthritis?

Research has identified autoantibodies against extracellular matrix proteins, including CLEC3A, in osteoarthritis patients. To study this autoimmune component:

Recommended protocol:

• Patient sample preparation:

  • Collect serum from OA patients and healthy donors

  • Use 10 patients minimum per group for statistical validity

• Detection method:

  • SDS-PAGE followed by immunoblotting

  • Use recombinant CLEC3A as positive control

• Cross-validation approach:

  • Test for concurrent autoantibodies against TSP-4 and COMP

  • Develop a panel approach for higher sensitivity

Expected findings pattern:

Research shows 9/10 OA patients exhibit IgG autoantibodies against at least one of these proteins (TSP-4, COMP, or CLEC3A) compared to only 1/10 healthy donors, suggesting potential utility as a diagnostic panel .

What are the critical quality control steps for validating CLEC3A antibodies in experimental systems?

Comprehensive validation of CLEC3A antibodies requires multiple quality control steps:

Pre-experimental validation:

• Epitope analysis:

  • Verify antibody targets unique CLEC3A regions with minimal homology to other C-type lectins

  • For human studies, antibodies targeting the C-terminal region sequence "GKFVDVNGIAISFLNWDRAQPNGGKRENCVLFSQSAQGKWSDEACRSSKR" show high specificity

• Cross-reactivity testing:

  • Test against protein array of 364 human recombinant protein fragments

  • Verify reactivity in multiple species if doing comparative studies

• Isotype verification:

  • Confirm antibody isotype (typically IgG) and purification method

Experimental validation:

• Positive controls:

  • Human cartilage tissue (high CLEC3A expression)

  • Chondrocytes or nucleus pulposus cells

• Negative controls:

  • Osteoclasts (very low CLEC3A expression)

  • Primary antibody omission control

  • Isotype control antibody

• Knockdown validation:

  • siRNA-mediated CLEC3A knockdown followed by antibody detection

  • Expected pattern: parallel decline in CLEC3A protein detection and chondrocyte marker genes like Col2a1

This validation approach ensures antibody specificity and significantly reduces false-positive results in CLEC3A research.

How can researchers use CLEC3A antibodies to investigate its role in tumor biology and chemosensitivity?

CLEC3A has emerging significance in tumor biology, particularly in osteosarcoma (OS) and pancreatic neuroendocrine tumors (PNETs). For investigating these relationships:

For osteosarcoma research:

• Expression analysis methodology:

  • Comparative immunoblotting of OS tissues vs. adjacent non-tumor tissues

  • Correlation with TNM stage and lymph node metastasis

• Functional investigation:

  • siRNA knockdown followed by:

    • Cell Counting Kit-8 assays for proliferation

    • Colony formation assays

    • Cell cycle distribution analysis

    • Treatment with chemotherapeutic agents (doxorubicin, cisplatin)

• Pathway analysis:

  • Monitor changes in AKT1/mTOR/HIF1α pathway components

  • Assess nuclear localization of HIF1α

  • Measure expression of HIF1α target genes (VEGF, GLUT1, MCL1)

For PNETs research:

• Recurrence prediction methodology:

  • Immunohistochemical scoring (0-6 scale)

  • Four-grade system based on staining intensity (0-3) and percentage of positive cells (0-3)

  • Positive expression threshold: score ≥3

• Application of CLEC3A antibody in tissue microarrays:

  • Dilution: 1:5000

  • Correlation with clinicopathological features and recurrence

Research has demonstrated that CLEC3A suppression enhances chemosensitivity to doxorubicin and cisplatin while inhibiting proliferation through the AKT1/mTOR/HIF1α pathway . In PNETs, positive CLEC3A expression significantly correlates with higher recurrence rates (P = 0.028) .

What considerations should researchers make when selecting between monoclonal and polyclonal CLEC3A antibodies for specific applications?

The choice between monoclonal and polyclonal CLEC3A antibodies should be application-driven:

Polyclonal antibodies advantages:

• Superior for detecting native proteins in tissues

• Better signal amplification in IHC applications (especially for low-abundance targets)

• More tolerant to fixation-induced epitope modifications

• Broader reactivity across species due to recognition of multiple epitopes

• Particularly effective for CLEC3A detection in paraffin-embedded tissues at dilutions of 1:5000-1:10000

Monoclonal antibodies advantages:

• Higher batch-to-batch consistency

• Reduced background in clean systems

• More suitable for quantitative analyses

• Better for distinguishing between closely related proteins

• Preferred for applications requiring absolute specificity

Application-specific recommendations:

The decision should be guided by the specific research question, with polyclonals generally preferred for discovery and monoclonals for validation or quantification studies.

How can researchers design experiments to investigate CLEC3A's role in intervertebral disc degeneration?

Investigating CLEC3A in intervertebral disc degeneration (IVDD) requires a multifaceted approach:

Tissue expression profiling:

• Compare CLEC3A expression in:

  • Healthy IVD from age-matched controls

  • Degenerated IVD tissues at various stages

  • Nucleus pulposus cells specifically

• Quantitative methods:

  • RT-qPCR for mRNA expression

  • Western blot for protein levels

  • Immunohistochemistry for spatial distribution (1:5000 dilution recommended)

Functional studies design:

• In vitro nucleus pulposus cell models:

  • CLEC3A overexpression via lentiviral vectors

  • CLEC3A knockdown via siRNA (at least two specific siRNAs recommended)

  • Assessment of:

    • Cell proliferation (via CCK-8)

    • Matrix accumulation (alcian blue staining)

    • Expression of chondrocyte marker genes (Col2a1)

• Signaling pathway investigation:

  • Monitor phosphorylation status of AKT

  • Apply pathway inhibitors (MK-2206 for PI3K-AKT)

  • Assess expression of cell cycle facilitating genes (PCNA, CCND1)

What approaches can researchers use to investigate potential therapeutic applications of CLEC3A in cartilage disorders?

Research indicates CLEC3A has therapeutic potential for cartilage disorders. To investigate this:

For osteoarthritis therapeutic research:

• Autoimmune component investigation:

  • Detection of autoantibodies against CLEC3A in OA patient serum

  • Testing immune suppressive therapy in OA patient subgroups with high autoantibody concentrations

  • Correlating autoantibody levels with disease progression

• CLEC3A-derived peptides approach:

  • Developing cartilage-specific antimicrobial peptides (AMPs) based on CLEC3A structure

  • Evaluating efficacy in septic arthritis models

  • Testing tissue specificity and antimicrobial properties

For intervertebral disc degeneration:

• Regenerative medicine applications:

  • Assessing CLEC3A's potential to enhance matrix accumulation

  • Investigating promotion of chondrogenesis in precursor cells

  • Testing CLEC3A-overexpressing cells in disc degeneration models

• Pathway modulation strategy:

  • Using CLEC3A to activate PI3K-AKT pathway in nucleus pulposus cells

  • Monitoring proliferation and matrix production

  • Evaluating long-term structural and functional outcomes

Experimental design considerations:

• Both in vitro and in vivo models required

• Combination with other growth factors (e.g., BMP2) for potential synergistic effects

• Assessment of potential side effects on osteoblastogenesis

Research has demonstrated that CLEC3A can facilitate chondrogenesis by accelerating cell proliferation and enhancing cartilage-specific ECM architecture, providing promising possibilities for clinical treatment of chondrocyte-related diseases and IVDD .

How should researchers approach troubleshooting when CLEC3A antibodies show inconsistent results in different experimental systems?

When encountering inconsistent CLEC3A antibody results across experimental systems, a systematic troubleshooting approach is essential:

Sample preparation issues:

• Tissue-specific optimization:

  • Different fixatives affect epitope preservation differently

  • For immunohistochemistry: citrate buffer (pH 6.0) heating at 100°C for 20 minutes recommended

  • For western blot: include both reducing and non-reducing conditions to detect potential dimers

• Expression level variations:

  • CLEC3A shows dramatic abundance differences between tissues:

    • High: cartilage, IVD, chondrocytes, NP cells

    • Low: osteoblasts, bone

    • Very rare: osteoclasts

Antibody selection factors:

• Epitope consideration:

  • Verify antibody recognizes native vs. denatured epitopes

  • C-terminal targeting antibodies show higher specificity

  • Check for potential post-translational modifications affecting binding

• Cross-reactivity evaluation:

  • Test against related C-type lectins (particularly CLEC3B)

  • For cross-species applications, check sequence homology:

    • Mouse/Human: 100% for some epitopes

    • Dog: 86%

    • Rabbit: 86%

    • Zebrafish: 79%

Protocol optimization matrix: