ADAMTS17 Antibody

What epitopes are targeted by commercial ADAMTS17 antibodies?

Commercial ADAMTS17 antibodies target various epitopes within the protein's structure. Rabbit polyclonal antibodies like ab198881 are typically raised against synthetic peptides within the human ADAMTS17 sequence . Mouse monoclonal antibodies may target specific domains - for example, the Q-12 antibody recognizes epitopes within ADAMTS17 that are conserved across mouse, rat, and human species . When selecting an antibody, researchers should consider which domain they need to detect, as some antibodies target the metalloprotease domain while others target the thrombospondin motifs or ancillary domains.

For validation studies, it's essential to know the precise epitope location, especially when studying different isoforms. ADAMTS17 has two identified isoforms: isoform a (22 exons) and isoform b (16 exons), with distinct expression patterns across tissues . An antibody targeting a region present in only one isoform will not detect the other.

What species reactivity can be expected with ADAMTS17 antibodies?

Based on published research, ADAMTS17 antibodies show varied species reactivity:

When working with non-human tissues, preliminary validation is recommended as antibody reactivity may vary based on epitope conservation. Recent studies have successfully used monoclonal ADAMTS17 antibodies to validate knockout mice, demonstrating specificity through reduced signal in ADAMTS17 KO and ADAMTS10/ADAMTS17 double knockout (DKO) tissues .

What is the subcellular localization pattern of ADAMTS17 when detected with antibodies?

ADAMTS17 exhibits specific localization patterns that vary by tissue type:

• In wild-type mouse skin and tibial growth plate, ADAMTS17 immunoreactivity appears around hair follicles and in hypertrophic chondrocytes, respectively

• In primary wild-type skin fibroblasts, the ADAMTS17 signal is most intense in perinuclear regions (corresponding to endoplasmic reticulum and Golgi) and in patches between cells, indicating secretion and extracellular matrix (ECM) association

• In intervertebral discs, immunohistochemistry confirms localized expression in the annulus fibrosus (AF) but minimal detection in the nucleus pulposus (NP)

Researchers should note that some studies have raised concerns about potential non-specific staining: "the reported localization of ADAMTS17 in the cytoplasm raises the possibility that the antibody used in this study was non-specific in tissue staining" . This emphasizes the importance of rigorous validation using knockout tissue controls.

How should ADAMTS17 antibodies be validated for specificity?

Rigorous validation is essential for ADAMTS17 antibodies due to potential cross-reactivity with other ADAMTS family members. The following methodological approach is recommended:

• Genetic validation: Use tissues or cells from ADAMTS17 knockout models as negative controls. Recent publications demonstrate this approach by showing reduced immunoreactivity in ADAMTS17 KO tissues compared to wild-type .

• Western blot validation: Verify detection of the correct molecular weight species. Full-length ADAMTS17 zymogen should appear at approximately 160 kDa (observed) or 125.3 kDa (predicted), while mature ADAMTS17 (after propeptide cleavage) should appear at approximately 130 kDa (observed) or 102.9 kDa (predicted) . The discrepancy between predicted and observed molecular weights is attributed to glycosylation.

• Recombinant protein controls: Use purified recombinant ADAMTS17 proteins or overexpression systems as positive controls.

• Empty vector controls: Demonstrate lack of reactive bands in conditioned medium and cell lysates from cells transfected with empty vectors .

• Multiple antibody validation: Use antibodies targeting different epitopes of ADAMTS17 (e.g., propeptide, metalloprotease domain, ancillary domain) to confirm specificity .

What are the recommended protocols for detecting ADAMTS17 by Western blot?

Based on published research protocols, the following methodology is recommended for Western blot detection of ADAMTS17:

Sample preparation:

• For cell lysates: Standard lysis buffers containing protease inhibitors are suitable

• For secreted ADAMTS17: Concentrate conditioned medium using appropriate methods (e.g., TCA precipitation or centrifugal filters)

Gel electrophoresis:

• Use 8% SDS-PAGE gels to adequately resolve the high molecular weight ADAMTS17 (120-160 kDa)

• Load sufficient protein (≥40 μg for cell lysates)

Transfer and detection:

• Use PVDF membrane for optimal protein binding

• Block with 5% non-fat milk or BSA

• Primary antibody dilutions:

  • Rabbit polyclonal (ab198881): 1/1300 dilution

  • Other antibodies: Follow manufacturer recommendations

• Secondary antibody: HRP-conjugated or fluorescent

• Detection: ECL technique with extended exposure (up to 1 hour may be necessary)

Expected results:

• Zymogen form: ~160 kDa

• Mature form (after propeptide cleavage): ~130 kDa

• Note: ADAMTS17 undergoes autoproteolysis, which may result in multiple bands

What are the optimal conditions for immunohistochemical detection of ADAMTS17?

For successful immunohistochemical detection of ADAMTS17 in tissues, researchers should consider the following protocol based on published studies:

For paraffin-embedded tissues:

• Deparaffinize and rehydrate sections using standard protocols

• Perform antigen retrieval (specific method may need optimization)

• Block endogenous peroxidase and non-specific binding

• Apply primary antibody:

  • Rabbit polyclonal (ab198881): 1/60 dilution

  • Monoclonal antibodies: 1/100 dilution

• Incubate at 4°C overnight or at room temperature for 1-2 hours

• Apply appropriate secondary antibody:

  • For fluorescent detection: Anti-mouse Alexa Fluor Plus 488 (1:1,000) or anti-rabbit Alexa Fluor Plus 488 (1:1,000)

  • For enzymatic detection: Anti-mouse or anti-rabbit HRP-conjugated (e.g., EnVision-Plus System-HRP)

• For enzymatic detection, develop using peroxidase substrate (3,3′-diaminobenzide) and counterstain with hematoxylin

Tissue-specific considerations:

• For skin sections: Focus on regions around hair follicles where ADAMTS17 is highly expressed

• For growth plate sections: Focus on hypertrophic chondrocytes

• For intervertebral disc sections: Examine the annulus fibrosus specifically, as ADAMTS17 expression is minimal in nucleus pulposus

How can researchers overcome problems with detecting secreted ADAMTS17?

Detecting secreted ADAMTS17 presents unique challenges due to its rapid autoproteolysis after secretion. Researchers have developed several strategies to address this issue:

• Use of catalytically inactive mutants: The ADAMTS17 EA mutant (Glu390Ala mutation in the active site) prevents autoproteolysis, making it easier to detect the secreted protein . This approach has been successfully used in multiple studies.

• Domain-specific antibodies: Employ antibodies targeting different domains to detect specific fragments. Studies have used antibodies against:

  • The propeptide domain

  • The ancillary domain

  • C-terminal tags (e.g., myc-tag)

• Concentrated conditioned media: Collect conditioned media at early time points after transfection and concentrate samples.

• Protease inhibitors: Add broad-spectrum protease inhibitors to the culture media, although this may not fully prevent ADAMTS17 autoproteolysis.

• Mass spectrometry approaches: For detection of ADAMTS17 fragments, N-terminomics strategies like Terminal Amine Isotopic Labeling of Substrates (TAILS) have been successfully employed .

How can ADAMTS17 antibodies be used to study protein-protein interactions?

ADAMTS17 antibodies can be valuable tools for investigating protein-protein interactions through multiple approaches:

• Co-immunoprecipitation (Co-IP): ADAMTS17 antibodies suitable for immunoprecipitation (such as Q-12 ) can be used to pull down ADAMTS17 along with its binding partners. This approach has helped identify interactions with:

  • Fibrillin-1 and Fibrillin-2

  • Extracellular matrix proteins like COL6A2, COL6A3, and fibronectin (FN1)

• Proximity ligation assays: These can detect protein interactions in situ using pairs of antibodies against ADAMTS17 and potential binding partners.

• Immunofluorescence co-localization: Double immunofluorescence staining with ADAMTS17 antibodies and antibodies against potential binding partners can provide evidence for co-localization in tissues.

• Validation of yeast-2-hybrid findings: Recent studies have used yeast-2-hybrid screening with the ADAMTS17 ancillary domain as bait to identify binding partners, including:

  • Thrombospondin-1 (THSB1)

  • Fibulin-3 (FBLN3)

  • ADAM12 and PAPPA

  • Extracellular domain of ERBB3

These interactions can be further validated using antibodies in co-IP or co-localization studies.

• Blocking antibodies: Developing function-blocking antibodies against specific ADAMTS17 domains can help determine which regions are critical for particular protein-protein interactions.

What are the challenges in differentiating between ADAMTS17 isoforms using antibodies?

Researchers face several challenges when attempting to distinguish between ADAMTS17 isoforms using antibodies:

• Isoform-specific epitopes: ADAMTS17 has two identified isoforms - isoform a (22 exons) and isoform b (16 exons) - with distinct expression patterns across tissues . Antibodies raised against shared regions cannot distinguish between these isoforms.

• Post-translational modifications: ADAMTS17 undergoes extensive post-translational modifications including:

  • N-glycosylation at seven sites, which affects protein stability and function

  • Propeptide cleavage by furin-like proteases

  • Autoproteolytic processing

• Detection strategies:

  • Western blotting with isoform-specific antibodies can distinguish isoforms based on size

  • RT-PCR using isoform-specific primers provides a complementary approach to verify antibody specificity

  • Mass spectrometry can identify isoform-specific peptides

• Validation approaches:

  • Use recombinant expression systems producing only one isoform

  • Create isoform-specific knockout models

  • Target unique exons in each isoform for antibody development

How can ADAMTS17 antibodies be used to study its autoproteolytic processing?

ADAMTS17's unusual life cycle involves rapid autoproteolytic processing, which can be studied using antibodies through the following approaches:

• Comparative analysis of wild-type and inactive mutants: Studies have demonstrated that while wild-type ADAMTS17 is poorly detected in conditioned medium due to autoproteolysis, the catalytically inactive ADAMTS17 EA mutant is readily detected . This comparative approach allows characterization of:

  • Processing kinetics

  • Cleavage sites

  • Fragment stability

• Domain-specific antibody panels: Using antibodies targeting different domains:

  • Anti-propeptide antibodies

  • Anti-metalloprotease domain antibodies

  • Anti-ancillary domain antibodies

  • C-terminal tag antibodies (e.g., myc-tag)

• Time-course experiments: Monitoring ADAMTS17 processing over time using pulse-chase experiments and immunoprecipitation with domain-specific antibodies.

• Trans-proteolysis studies: Research has shown ADAMTS17 can undergo autocatalytic processing in trans. This can be studied using co-culture systems with:

  • Wild-type ADAMTS17

  • ADAMTS17 EA mutant

  • ADAMTS17 ancillary domain constructs (ADAMTS17-AD)

• Fragment identification: Mass spectrometry paired with immunoprecipitation using domain-specific antibodies can identify precise cleavage sites and processing intermediates.

What experimental designs effectively demonstrate ADAMTS17 antibody specificity?

Demonstrating ADAMTS17 antibody specificity is critical for research integrity. The following experimental designs provide robust validation:

• Genetic knockout controls:

  • The most definitive approach uses ADAMTS17 knockout tissues/cells

  • Recent studies validated monoclonal ADAMTS17 antibodies using:

    • ADAMTS17 KO mice

    • ADAMTS10/ADAMTS17 double knockout (DKO) mice

  • Immunostaining should show strong reduction or absence of signal in knockout samples

• CRISPR/Cas9-engineered cell lines:

  • Create ADAMTS17-deficient cell lines using CRISPR/Cas9 genome editing

  • Compare antibody reactivity between wild-type and knockout cells

• siRNA/shRNA knockdown:

  • Reduce ADAMTS17 expression through RNA interference

  • Demonstrate corresponding reduction in antibody signal

  • Include non-targeting controls

• Recombinant expression systems:

  • Overexpress ADAMTS17 in cells with low endogenous expression

  • Show increased antibody signal proportional to expression level

  • Include empty vector controls

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide

  • Demonstrate blocked antibody binding in Western blot or immunohistochemistry

• Cross-reactivity assessment:

  • Test antibody against related ADAMTS family proteins

  • Particularly important for distinguishing ADAMTS17 from ADAMTS10, which shares functional redundancy

Why might Western blots with ADAMTS17 antibodies show multiple bands?

Multiple bands in ADAMTS17 Western blots are common and may result from several biological and technical factors:

• Autoproteolytic processing: ADAMTS17 undergoes extensive autocatalytic processing after secretion, generating multiple fragments. Research has shown that:

  • The zymogen form appears at ~160 kDa (observed) or 125.3 kDa (predicted)

  • The mature form after propeptide cleavage appears at ~130 kDa (observed) or 102.9 kDa (predicted)

  • Additional fragments may appear depending on the antibody's epitope location

• Post-translational modifications:

  • Variable glycosylation: ADAMTS17 contains seven N-glycosylation sites

  • Differential glycosylation across cell types can alter apparent molecular weight

  • Deglycosylation experiments (using PNGase F) can help identify glycosylation-dependent mobility shifts

• Isoforms and splice variants:

  • ADAMTS17 has two identified isoforms (a and b)

  • Additional tissue-specific splice variants may exist

• Partial degradation:

  • Sample preparation without adequate protease inhibitors may result in degradation

  • Freeze-thaw cycles can promote degradation

• Cross-reactivity:

  • Antibodies may cross-react with other ADAMTS family members

  • Validate specificity using knockout controls or peptide competition assays

How can researchers optimize immunohistochemical detection of ADAMTS17 in different tissues?

Optimizing ADAMTS17 immunohistochemistry requires tissue-specific considerations:

• Fixation protocols:

  • For most tissues, 4% paraformaldehyde fixation works well

  • Overfixation can mask epitopes; optimize fixation time

  • For bone or cartilage, decalcification protocols must preserve antigenicity

• Antigen retrieval methods:

  • Heat-induced epitope retrieval: Test citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0)

  • Enzymatic retrieval: May be necessary for heavily fixed tissues

  • For growth plate tissues, mild enzymatic treatment (hyaluronidase) may improve accessibility

• Antibody optimization by tissue type:

• Signal amplification strategies:

  • Tyramide signal amplification for low-abundance expression

  • Polymer-based detection systems (e.g., EnVision-Plus System-HRP)

  • Fluorescent secondary antibodies with appropriate filters to reduce autofluorescence

• Background reduction:

  • Extended blocking (2+ hours) with serum matching secondary antibody host

  • Addition of 0.1-0.3% Triton X-100 for improved antibody penetration

  • Avidin/biotin blocking for tissues with high biotin content

What are the critical factors for successful co-immunoprecipitation with ADAMTS17 antibodies?

Successful co-immunoprecipitation (co-IP) of ADAMTS17 and its binding partners requires attention to several critical factors:

• Antibody selection:

  • Verify the antibody is suitable for immunoprecipitation (e.g., Q-12 antibody)

  • Choose antibodies targeting domains away from interaction interfaces

  • Consider using tagged recombinant ADAMTS17 and anti-tag antibodies

• Lysis conditions:

  • Use mild lysis buffers to preserve protein-protein interactions

  • Typical buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40 or 0.5% Triton X-100

  • Include protease inhibitors to prevent degradation

  • Add phosphatase inhibitors if studying phosphorylation-dependent interactions

• Cross-linking considerations:

  • For transient interactions, consider chemical cross-linkers like DSP or formaldehyde

  • Cross-linking can stabilize ADAMTS17 complexes before lysis

• Pre-clearing step:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Use isotype control antibodies as negative controls

• Detection strategies:

  • For ADAMTS17-substrate interactions, use domain-specific antibodies

  • For detection of autoproteolytic fragments, use antibodies against different domains

  • Mass spectrometry can identify novel binding partners

• Special considerations for secreted ADAMTS17:

  • Concentrate conditioned media before immunoprecipitation

  • Use the inactive ADAMTS17 EA mutant to prevent autoproteolysis

  • Consider extracellular matrix components that may co-precipitate

How can researchers investigate ADAMTS17 expression in tissues with low abundance?

Detecting low-abundance ADAMTS17 in tissues requires sensitive methodological approaches:

• Signal amplification in immunohistochemistry:

  • Tyramide signal amplification (TSA) can increase sensitivity 10-100 fold

  • Multi-step detection systems (e.g., biotin-streptavidin)

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

• RT-qPCR validation:

  • Complement protein detection with mRNA analysis

  • Design primers spanning exon-exon junctions

  • Use digital droplet PCR for absolute quantification of rare transcripts

  • Reference gene validation is critical for tissues with variable expression

• Enrichment strategies:

  • Laser capture microdissection to isolate specific cell populations

  • Immunoprecipitation followed by Western blotting

  • Proximity ligation assay (PLA) for in situ detection

• Single-cell approaches:

  • Single-cell RNA sequencing to identify cell populations expressing ADAMTS17

  • Flow cytometry with intracellular staining for quantitative analysis

• Considerations for specific tissues:

  • For intervertebral discs: Focus on annulus fibrosus (AF) where ADAMTS17 is expressed rather than nucleus pulposus (NP) where expression is minimal

  • For eye tissues: Concentrate on lens equator and trabecular region where expression is higher

  • For growth plates: Focus on hypertrophic chondrocytes

What controls are essential when using ADAMTS17 antibodies in functional studies?

For functional studies involving ADAMTS17 antibodies, the following controls are essential:

• Genetic controls:

  • ADAMTS17 knockout tissues/cells as negative controls

  • Rescue experiments with recombinant ADAMTS17 expression

  • ADAMTS17 domain deletion mutants to map functional regions

• Antibody specificity controls:

  • Isotype-matched control antibodies

  • Antibody pre-absorption with immunizing peptide

  • Multiple antibodies targeting different ADAMTS17 epitopes

• Functional validation controls:

  • Catalytically inactive ADAMTS17 EA mutant (Glu390Ala)

  • Domain-specific blocking antibodies

  • Small molecule inhibitors as complementary approach

• Expression level controls:

  • Dose-dependent antibody effects

  • Correlation between antibody binding and functional outcomes

  • Time-course experiments to establish causality

• Context-specific controls:

  • For studies of ADAMTS17's role in BMP-Smad1/5/8 pathway: Include BMP pathway inhibitors

  • For fibrillin interaction studies: Include fibrillin-1 and fibrillin-2 knockdown controls

  • For substrate studies: Include known ADAMTS family substrates as positive controls

• Reproducibility considerations:

  • Multiple antibody lots

  • Different cell lines or tissue sources

  • Alternative methodological approaches to confirm findings