ADAMTS16 Antibody

What is ADAMTS16 and why is it important in research?

ADAMTS16 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 16) is a member of the ADAMTS family of secreted proteinases. This protein has emerged as a critical regulator in multiple physiological and pathological processes. Research has shown that ADAMTS16 plays crucial roles in:

• Cardiac fibrosis through activation of latent TGF-β

• Blood pressure regulation via mechanisms involving TGF-β signaling

• Epithelial-mesenchymal transition (EMT) and cancer metastasis, particularly in lung adenocarcinoma

• Extracellular matrix remodeling and tissue development

The importance of ADAMTS16 in these diverse pathways makes antibodies against this protein valuable tools for investigating disease mechanisms and potential therapeutic targets.

What are the key applications for ADAMTS16 antibodies in research?

ADAMTS16 antibodies have several validated research applications:

These applications allow researchers to detect, quantify, and localize ADAMTS16 in various experimental settings, facilitating investigations into its expression patterns and functional roles .

How is the expression of ADAMTS16 distributed in different tissues?

Studies have shown tissue-specific expression patterns for ADAMTS16:

• Kidney: Prominently expressed, making it relevant for blood pressure regulation studies

• Heart: Expressed and upregulated in cardiac hypertrophy and heart failure models

• Lung: Detected in lung tissue and implicated in lung adenocarcinoma progression

• Other tissues: Also detected in thymus, liver, and spleen at varying levels

This tissue distribution pattern informs the selection of appropriate positive control samples for antibody validation and experimental design.

How should I select the most appropriate ADAMTS16 antibody for my specific research application?

Selection criteria should include:

• Epitope consideration: Determine which domain of ADAMTS16 is most relevant to your research question. Some antibodies target the N-terminal region while others target C-terminal domains. For instance, antibodies raised against synthetic peptides within human ADAMTS16 may recognize different epitopes .

• Validated applications: Confirm that the antibody has been validated for your specific application:

  • For signaling pathway studies involving TGF-β, select antibodies validated in co-immunoprecipitation

  • For expression studies, ensure validation in Western blot or immunohistochemistry

• Species reactivity: Verify cross-reactivity with your experimental model organism. Most commercial ADAMTS16 antibodies react with human and mouse samples .

• Published validation: Review literature citations where the antibody has been successfully used. For example, ab45048 has been cited in 5 publications according to search result .

What controls should I include when using ADAMTS16 antibodies?

A robust experimental design should include:

• Positive controls:

  • PC3 cell lysates have been documented to express ADAMTS16

  • Mouse lung mesenchymal MLg cells, particularly when treated with specific stimuli like TPA or IL1-alpha

  • Kidney tissue samples, given the high expression of ADAMTS16 in this organ

• Negative controls:

  • Samples with ADAMTS16 knockdown using siRNA, as demonstrated in mouse cardiac fibroblasts (MCFs)

  • Tissues from Adamts16 knockout rats with a 17 bp deletion in exon 1

  • Secondary antibody-only controls to rule out non-specific binding

• Specificity controls:

  • Blocking peptide experiments using the immunizing peptide

  • Comparison of results with multiple antibodies targeting different epitopes of ADAMTS16

How can I optimize Western blot conditions for ADAMTS16 detection?

Based on published research protocols:

• Gel percentage selection: Use 8% SDS-PAGE gels, as documented in successful detection protocols .

• Sample preparation considerations:

  • Load adequate protein (40 μg of total protein is commonly used)

  • For comprehensive analysis, fractionate samples to analyze cell lysate, conditioned medium, and extracellular matrix separately, as ADAMTS16 distribution varies between these fractions

• Antibody dilution optimization:

  • Start with recommended dilutions (e.g., 1:1000-1:1350 for ab45048 and ab198917)

  • Consider extended exposure times (30 minutes has been documented as effective)

• Band pattern interpretation:

  • Be aware that ADAMTS16 can appear as multiple bands:

    • Full-length protein at approximately 136 kDa

    • Multiple splice variants (151.38 kDa, 136.2 kDa, 119.5 kDa)

    • Shorter versions (62 kDa and 56 kDa) lacking the MP domain

  • A doublet pattern around 130 kDa has been observed in some preparations

How do I resolve discrepancies between predicted and observed molecular weights of ADAMTS16?

ADAMTS16 presents complex banding patterns that require careful interpretation:

• Expected molecular weight variations:

  • The full-length ADAMTS16 polypeptide has a predicted molecular weight of 136 kDa

  • Several splice variants have been reported: 151.38 kDa, 136.2 kDa, and 119.5 kDa

  • Two shorter versions (62 kDa and 56 kDa) lacking the metalloproteinase domain have been documented

• Post-translational modifications:

  • As a metalloproteinase, ADAMTS16 undergoes proteolytic processing

  • Glycosylation can increase apparent molecular weight

  • The presence of thrombospondin motifs can affect protein migration

• Sample preparation effects:

  • Different extraction methods may preserve or disrupt protein complexes

  • ADAMTS16 has been shown to distribute differently between cell lysate, conditioned medium, and extracellular matrix fractions

• Resolution strategies:

  • Compare results with multiple antibodies targeting different epitopes

  • Include positive control samples with known expression patterns

  • Consider using knockout/knockdown samples as negative controls to confirm specificity

How should I interpret contradictory findings about ADAMTS16 function in different disease models?

ADAMTS16 exhibits context-dependent functions that may appear contradictory:

• In cardiac pathology:

  • ADAMTS16 activates latent TGF-β, promoting cardiac fibrosis and contributing to heart failure

  • The RRFR motif in ADAMTS16 mediates interaction with LAP-TGF-β, suggesting a specific molecular mechanism

• In hypertension models:

  • Targeted disruption of the Adamts16 gene (17 bp deletion in exon 1) in rats decreases blood pressure by 36 mmHg

  • This effect may be mediated through inhibition of TGF-β activation, as knockout of Adamts16 inhibits TGF-β activation

• In cancer models:

  • ADAMTS16 drives EMT and metastasis in lung adenocarcinoma through a positive feedback loop with the TGF-β1/SOX4 axis

  • ADAMTS16 can serve as both a prognostic biomarker and potential therapeutic target

• Reconciliation approach:

  • Consider tissue-specific regulatory mechanisms

  • Evaluate experimental model differences (genetic knockout vs. knockdown)

  • Examine the specific ADAMTS16 domains involved in each context

  • Focus on the common TGF-β pathway as a unifying mechanism across different disease models

What are the potential cross-reactivity concerns with ADAMTS16 antibodies?

Consider these factors when evaluating antibody specificity:

• Homology with other ADAMTS family members:

  • ADAMTS16 has high sequence similarity to ADAMTS18, potentially leading to cross-reactivity

  • The conservation of the disintegrin-like domain and thrombospondin motifs across the ADAMTS family necessitates careful epitope selection

• Validation strategies to ensure specificity:

  • Use siRNA knockdown experiments to confirm signal reduction

  • Compare results from antibodies targeting different epitopes

  • Include tissues from Adamts16 knockout animals when available

  • Perform peptide competition assays with the immunizing peptide

• Species-specific considerations:

  • While many antibodies are raised against human ADAMTS16, cross-reactivity with mouse and rat orthologs should be verified

  • Sequence alignment analysis between species can help predict potential cross-reactivity issues

How can ADAMTS16 antibodies be utilized to investigate TGF-β activation mechanisms?

Research has established ADAMTS16 as a novel regulator of TGF-β activation:

• Co-immunoprecipitation (Co-IP) approach:

  • Use ADAMTS16 antibodies to immunoprecipitate protein complexes

  • Western blot for LAP-TGF-β to confirm interaction

  • Compare wild-type ADAMTS16 with RRFR-to-IIFI mutant to study binding specificity

• Functional activation assays:

  • Monitor LAP-TGF-β cleavage in the presence of ADAMTS16

  • Measure active TGF-β levels using ELISA in control vs. ADAMTS16-overexpressing cells

  • Include TGF-β neutralizing antibody to confirm the specificity of observed effects

• Combined knockdown and rescue experiments:

  • Use siRNA to knock down Adamts16 expression

  • Assess LAP-TGF-β levels and TGF-β-dependent transcription activation

  • Perform rescue experiments with wild-type vs. mutant ADAMTS16

This methodology has revealed that the RRFR motif of ADAMTS16 is critical for its interaction with LAP-TGF-β and subsequent TGF-β activation .

What methodological approaches can be used to study ADAMTS16 in metastasis and EMT?

Based on recent research in lung adenocarcinoma:

• Transcriptome analysis correlation:

  • Analyze correlation between ADAMTS16 expression and EMT markers

  • Use tissue microarrays and immunohistochemistry with ADAMTS16 antibodies to validate expression patterns

  • Correlate expression with patient outcomes to assess prognostic value

• In vitro functional assays:

  • Manipulate ADAMTS16 expression (knockdown/overexpression)

  • Assess migration abilities of cancer cells

  • Monitor EMT marker expression through Western blot and immunofluorescence

• In vivo metastasis models:

  • Establish animal models with modified ADAMTS16 expression

  • Monitor lung and pleural metastasis development

  • Perform histological analysis using ADAMTS16 antibodies

• Mechanistic investigation:

  • Employ ELISA and Western blot to assess TGF-β signaling activation

  • Perform co-IP to identify ADAMTS16 protein interactions

  • Use ChIP and dual-luciferase reporter assays to study transcriptional regulation

How can ADAMTS16 antibodies be used to investigate its role in cardiovascular pathophysiology?

Research has implicated ADAMTS16 in cardiac remodeling and blood pressure regulation:

• Expression analysis in disease models:

  • Use Western blot and qRT-PCR to assess ADAMTS16 upregulation in cardiac hypertrophy and heart failure models

  • Correlate expression with markers of extracellular matrix remodeling (Mmp2, Mmp9, Col1a1, Col3a1)

• Cellular localization studies:

  • Perform immunohistochemistry on cardiac tissue sections

  • Co-stain with markers of cardiac fibroblasts and myofibroblasts

  • Compare expression patterns in normal versus pressure-overloaded hearts

• Gain and loss of function approaches:

  • Study the effects of ADAMTS16 overexpression on cardiac fibroblast activation

  • Use genetic models with Adamts16 disruption to assess blood pressure phenotypes

  • Monitor the impact on TGF-β activation and downstream SMAD2/SMAD3 signaling

• Therapeutic intervention studies:

  • Test RRFR tetrapeptide effects on ADAMTS16-LAP-TGF-β interaction

  • Evaluate the impact on cardiac fibrosis and hypertrophy

  • Combine with TGF-β-neutralizing antibody to confirm specificity

What is the optimal protocol for validating a new ADAMTS16 antibody?

A comprehensive validation approach should include:

• Western blot validation:

  • Test against recombinant ADAMTS16 protein

  • Evaluate in tissues/cells with known expression (kidney, PC3 cells)

  • Compare with knockout/knockdown samples

  • Assess multiple fractions (cell lysate, conditioned medium, extracellular matrix)

• Peptide competition assay:

  • Pre-incubate antibody with the immunizing peptide

  • Compare signal with and without peptide competition

  • Include gradient concentrations of competing peptide

• Cross-reactivity assessment:

  • Test against closely related family members (especially ADAMTS18)

  • Compare results across multiple species if claiming cross-reactivity

  • Evaluate antibody performance in overexpression systems

• Functional validation:

  • Confirm ability to immunoprecipitate native ADAMTS16

  • Test in downstream applications (immunohistochemistry, ELISA)

  • Validate in relevant biological contexts (e.g., TGF-β activation assays)

How should researchers approach the detection of different ADAMTS16 isoforms?

Multiple approaches are necessary to comprehensively detect and characterize ADAMTS16 isoforms:

• Transcript analysis:

  • Use PCR with primers spanning different exon combinations

  • Validate transcript predictions from databases (e.g., NCBI vs. Ensembl)

  • Sequence-confirm amplified products as demonstrated in rat studies

• Protein isoform detection:

  • Select antibodies targeting preserved regions across isoforms

  • Use gradient gels to resolve closely migrating isoforms

  • Be aware of reported isoforms (151.38 kDa, 136.2 kDa, 119.5 kDa, 62 kDa, 56 kDa)

• Cellular distribution analysis:

  • Fractionate samples to analyze cell lysate, conditioned medium, and extracellular matrix separately

  • Different isoforms may preferentially localize to specific cellular compartments

• Functional characterization:

  • Compare activities of different isoforms (e.g., TGF-β activation capacity)

  • Assess domain-specific functions (especially for isoforms lacking specific domains)

What methodological approaches can detect post-translational modifications of ADAMTS16?

To characterize post-translational modifications:

• Glycosylation analysis:

  • Treat samples with glycosidases (PNGase F, Endo H)

  • Compare migration patterns before and after treatment

  • Look for mobility shifts indicating removal of glycans

• Proteolytic processing detection:

  • Use antibodies targeting different domains to detect processing events

  • Compare results between cell lysates and secreted fractions

  • Include protease inhibitors during sample preparation to prevent artifactual processing

• Phosphorylation studies:

  • Immunoprecipitate ADAMTS16 and probe with phospho-specific antibodies

  • Use phosphatase treatment to confirm specificity

  • Consider mass spectrometry for comprehensive phosphorylation site mapping

• Other modifications:

  • Investigate potential ubiquitination or SUMOylation through immunoprecipitation followed by specific antibody detection

  • Assess disulfide bond formation through non-reducing versus reducing conditions

How can ADAMTS16 antibodies facilitate research into its role as a therapeutic target?

Emerging research suggests ADAMTS16 as a potential therapeutic target:

• Target validation approaches:

  • Use antibodies to monitor ADAMTS16 expression in disease versus normal tissues

  • Correlate expression levels with disease progression and outcomes

  • Evaluate changes in expression following therapeutic interventions

• Functional blocking studies:

  • Develop and test function-blocking antibodies targeting the RRFR motif

  • Compare with RRFR tetrapeptide inhibition of ADAMTS16-LAP-TGF-β interaction

  • Assess functional outcomes in relevant disease models

• Biomarker development:

  • Evaluate ADAMTS16 as a prognostic biomarker in cancer

  • Develop sensitive ELISA protocols using validated antibody pairs

  • Correlate levels with disease progression and treatment response

• Combination therapy approaches:

  • Test ADAMTS16 inhibition in combination with TGF-β pathway modulators

  • Monitor effects on disease progression in animal models

  • Use antibodies to confirm target engagement

What are the methodological considerations for investigating ADAMTS16 in single-cell analyses?

As single-cell technologies advance, several approaches can be applied:

• Single-cell immunostaining:

  • Optimize ADAMTS16 antibody dilutions for immunocytochemistry

  • Combine with markers of cell identity and activation state

  • Use confocal microscopy to assess subcellular localization

• Mass cytometry (CyTOF) applications:

  • Metal-conjugate ADAMTS16 antibodies for CyTOF analysis

  • Combine with panels of cell type-specific and signaling markers

  • Analyze heterogeneity in expression across cell populations

• In situ hybridization with protein co-detection:

  • Combine RNAscope for ADAMTS16 transcript with antibody detection of protein

  • Assess correlation between transcript and protein levels at single-cell resolution

  • Identify cell types expressing ADAMTS16 in complex tissues

• Spatial transcriptomics integration:

  • Validate transcriptomic findings with antibody-based protein detection

  • Map ADAMTS16 expression to specific tissue microenvironments

  • Correlate with expression of interacting partners (e.g., TGF-β)