ADAMTS19 Antibody

What is ADAMTS19 and what are its biological functions?

ADAMTS19 (ADAM Metallopeptidase with Thrombospondin Type 1 Motif, 19) is a member of the ADAMTS family of secreted enzymes involved in extracellular matrix organization and tissue remodeling. In normal tissues, ADAMTS19 appears to play important roles in maintaining cellular homeostasis and tissue integrity. Recent evidence indicates that ADAMTS19 functions as a tumor suppressor in gastric cancer by inhibiting cell migration and invasion through interaction with the NF-κB pathway . Expression analysis shows that ADAMTS19 is significantly downregulated in gastric cancer tissues compared to adjacent normal tissues, with this downregulation correlating with poorer prognosis . These findings suggest ADAMTS19 may have broader implications in cancer biology and progression.

What types of ADAMTS19 antibodies are currently available for research applications?

Several types of ADAMTS19 antibodies are available for research purposes, primarily polyclonal antibodies derived from rabbit hosts. These include:

• Antibodies targeting the internal region of ADAMTS19 (e.g., ABIN6257740)

• Antibodies targeting specific amino acid sequences (e.g., AA 325-686, AA 298-328)

• Antibodies targeting the N-terminal region

Most available antibodies are unconjugated and show reactivity to human and mouse ADAMTS19, with predicted cross-reactivity to other species including pig, bovine, horse, sheep, and dog . These antibodies are purified using peptide affinity chromatography with SulfoLink™ Coupling Resin to ensure specificity .

What experimental applications are ADAMTS19 antibodies suitable for?

ADAMTS19 antibodies have demonstrated utility in multiple experimental applications:

The antibodies can detect endogenous levels of total ADAMTS19, making them suitable for studying native expression without requiring overexpression systems .

How should I optimize immunohistochemistry protocols for ADAMTS19 detection in tissue microarrays?

For optimal ADAMTS19 detection in tissue microarrays and histological specimens, follow this validated protocol:

• Use a biotin-streptavidin horseradish peroxidase (HRP) detection system

• Incubate tissue sections with primary rabbit antibodies against ADAMTS19 (ab190073, Abcam) at 1:1000 dilution at 4°C overnight

• Wash and incubate with appropriate secondary antibodies

• Develop with diaminobenzidine and counterstain with hematoxylin

• Use primary antibody diluent as a negative control

• Have two independent pathologists score ADAMTS19 expression

• Determine optimal cutoff scores using statistical software (X-tile software was used to determine a cutoff score of 5.5 for ADAMTS19 in gastric cancer studies)

This protocol has been successfully implemented in studies examining ADAMTS19 expression in gastric cancer tissue specimens .

What controls are essential when designing experiments involving ADAMTS19 antibodies?

Rigorous experimental design requires appropriate controls:

• Negative controls:

  • For IHC/ICC: Primary antibody diluent without antibody

  • For WB: Lysates from tissues/cells with confirmed low ADAMTS19 expression

• Positive controls:

  • For IHC: Normal gastric mucosa (shown to express higher levels of ADAMTS19 than cancer tissue)

  • For WB: Recombinant ADAMTS19 protein or lysates from tissues with confirmed expression

• Reference controls:

  • For qRT-PCR: GAPDH (with primers 5-GACAGTCAGCCGCATCTTCTT-3 forward and 5-AATCCGTTGACTCCGACCTTC-3 reverse)

  • For protein loading: Housekeeping proteins (β-actin, GAPDH)

• Validation controls:

  • Comparison of protein detection with mRNA expression analysis

  • Use of multiple antibodies targeting different epitopes when possible

How can I quantitatively analyze ADAMTS19 expression in comparative studies?

For robust quantitative analysis of ADAMTS19 expression, multiple approaches should be employed:

• For protein expression (IHC):

  • Use standardized scoring systems with multiple independent observers

  • Employ digital image analysis for objective quantification

  • Determine optimal cutoff values using statistical software like X-tile

  • Express results as mean ± standard deviation for statistical comparison

• For mRNA expression (qRT-PCR):

  • Use ADAMTS19-specific primers: 5-AGGCCAGTAACTGCTTGCTAC-3 (forward) and 5-GTCTAGCTTGGTTCTGCATTCTT-3 (reverse)

  • Normalize to reference genes (GAPDH recommended)

  • Calculate relative expression using the 2^(-ΔΔCt) method

• Statistical analysis:

  • Compare continuous variables using Student's t-test

  • Assess categorical variables using chi-squared or Wilcoxon signed-rank tests

  • Calculate survival using Kaplan-Meier method with log-rank test

  • Perform multivariate analysis using Cox proportional hazards regression

What is the functional significance of ADAMTS19 downregulation in gastric cancer?

ADAMTS19 downregulation appears to be a significant event in gastric carcinogenesis:

How does ADAMTS19 expression correlate with clinicopathological characteristics in cancer?

ADAMTS19 expression shows significant associations with several clinicopathological parameters in gastric cancer:

Interestingly, ADAMTS19 expression did not significantly correlate with age, gender, histologic type, differentiation, T stage (invasion depth), N stage (lymph node metastasis), TNM stage, or vessel invasion . Univariate analysis identified ADAMTS19 expression as a prognostic indicator, although multivariate analysis did not confirm it as an independent prognostic marker .

What molecular mechanisms underlie ADAMTS19's tumor-suppressive function?

The tumor-suppressive effects of ADAMTS19 in gastric cancer involve several molecular pathways:

• S100A16 targeting:

  • ADAMTS19 modulates the expression and/or activity of S100A16, a calcium-binding protein

  • This interaction appears critical for ADAMTS19's effects on cell migration and invasion

• NF-κB pathway regulation:

  • Co-immunoprecipitation assays demonstrate interaction between ADAMTS19 and P65 (a component of the NF-κB pathway)

  • This interaction suggests ADAMTS19 may directly modulate NF-κB signaling, which is known to control genes involved in cancer progression

• Protein-protein interactions:

  • The research protocol utilized specific antibodies (ab190073, Abcam for ADAMTS19; #8242, Cell Signaling Technology for P65) to study these interactions

  • These studies were conducted using stably overexpressed ADAMTS19 and control MGC803 cells

• Downstream effectors:

  • While not fully elucidated in the available data, the mechanism likely involves alteration of genes controlling cellular motility and invasive properties through NF-κB-dependent transcriptional regulation

How can I optimize western blotting protocols for reliable ADAMTS19 detection?

For reliable ADAMTS19 detection by western blotting:

• Sample preparation:

  • Use appropriate lysis buffers that preserve metalloprotease activity

  • Include protease inhibitors to prevent degradation

  • Denature samples at appropriate temperatures (typically 95°C for 5 minutes)

• Gel selection and transfer:

  • Use 8-10% SDS-PAGE gels to properly resolve ADAMTS19 (a relatively large protein)

  • Optimize transfer conditions for high molecular weight proteins

  • Consider using PVDF membranes for optimal protein retention

• Antibody selection and dilution:

  • Primary antibody: Use antibodies targeting the internal region of ADAMTS19 at 1:1000 dilution

  • Secondary antibody: HRP-conjugated anti-rabbit at 1:5000-1:10000

  • Incubate primary antibody overnight at 4°C for optimal results

• Controls and validation:

  • Include positive control lysates from tissues known to express ADAMTS19

  • Use GAPDH or β-actin as loading controls

  • For antibody validation, compare results with ADAMTS19 mRNA expression data

What protocol should I follow for co-immunoprecipitation studies involving ADAMTS19?

For co-immunoprecipitation studies investigating ADAMTS19 interactions:

• Cell preparation:

  • Generate cells stably overexpressing ADAMTS19 alongside appropriate controls

  • MGC803 cells have been successfully used in previous studies

• Lysis procedure:

  • Lyse cells using Pierce IP Lysis Buffer (#87788, Thermo Fisher Scientific)

  • Centrifuge cell supernatants at 4°C to remove debris

• Pre-clearing and immunoprecipitation:

  • Add 50 μL of protein A/G agarose bead solution to every 100 μL of cell supernatant

  • Use ADAMTS19 antibody (ab190073, Abcam) to pull down ADAMTS19 and interacting proteins

  • For reverse co-IP, use antibodies against potential interaction partners (e.g., P65 antibody #8242, Cell Signaling Technology)

  • Incubate overnight at 4°C with gentle rotation

• Controls and detection:

  • Use normal IgG as a negative control

  • Perform western blotting on immunoprecipitated material

  • Probe membranes with antibodies against both ADAMTS19 and suspected interaction partners

What are common challenges in qRT-PCR analysis of ADAMTS19 expression and how can they be overcome?

Researchers commonly encounter these challenges when analyzing ADAMTS19 expression by qRT-PCR:

• RNA quality and integrity:

  • Use TRIzol reagent (Invitrogen) for tissue samples or RNA-Quick Purification Kit (ES-RN001, Yishan Biotechnology) for cell samples

  • Verify RNA integrity by gel electrophoresis or Bioanalyzer before proceeding

• Primer design and validation:

  • Use validated ADAMTS19 primers: 5-AGGCCAGTAACTGCTTGCTAC-3 (forward) and 5-GTCTAGCTTGGTTCTGCATTCTT-3 (reverse)

  • Ensure primers span exon-exon junctions to avoid genomic DNA amplification

  • Validate primer specificity by melt curve analysis and agarose gel electrophoresis

• Reference gene selection:

  • GAPDH has been validated as a suitable reference gene: 5-GACAGTCAGCCGCATCTTCTT-3 (forward) and 5-AATCCGTTGACTCCGACCTTC-3 (reverse)

  • Consider using multiple reference genes for more robust normalization

• Data analysis and interpretation:

  • Use the 2^(-ΔΔCt) method for relative quantification

  • Present data as means ± standard deviations

  • Apply appropriate statistical tests (Student's t-test for comparing two groups)

How might ADAMTS19 expression analysis be integrated with other biomarkers for improved cancer prognostication?

Integration of ADAMTS19 with other biomarkers offers promising avenues for enhanced prognostication:

• Multi-marker panels:

  • Combine ADAMTS19 with established prognostic markers in gastric cancer

  • Integrate with S100A16 expression data (already shown to have a functional relationship with ADAMTS19)

  • Create predictive models incorporating both protein markers and clinicopathological features

• Molecular classification systems:

  • Include ADAMTS19 in molecular classification schemes for gastric cancer

  • Stratify patients based on combined expression patterns of multiple markers

  • The study already demonstrated patient stratification into four groups based on ADAMTS19 and S100A16 expression levels

• Integrated 'omics approaches:

  • Correlate ADAMTS19 expression with genomic, transcriptomic, and proteomic data

  • Identify molecular signatures that include ADAMTS19 for improved prediction

  • Explore relationships between ADAMTS19 expression and mutation profiles or microsatellite instability status

• Liquid biopsy development:

  • Explore potential for detecting ADAMTS19 in circulation as part of liquid biopsy approaches

  • Investigate correlation between tissue and circulating levels of ADAMTS19

What epigenetic mechanisms regulate ADAMTS19 expression in cancer and how can they be studied?

The negative correlation between ADAMTS19 expression and promoter methylation suggests important epigenetic regulatory mechanisms:

• Methylation analysis techniques:

  • Bisulfite sequencing of the ADAMTS19 promoter region

  • Methylation-specific PCR to quantify methylation levels

  • Genome-wide methylation arrays to identify specific CpG sites involved

• Functional studies:

  • Treatment of cancer cells with demethylating agents (5-aza-2'-deoxycytidine) to restore ADAMTS19 expression

  • CRISPR-based epigenome editing to specifically modulate methylation at the ADAMTS19 promoter

  • Correlation analysis between ADAMTS19 expression and DNA methyltransferase levels

• Histone modification analysis:

  • ChIP-seq to identify histone modifications at the ADAMTS19 locus

  • Investigate the role of histone deacetylases and methyltransferases in regulating ADAMTS19

  • Correlate findings with DNA methylation data for comprehensive epigenetic profiling

• Transcriptional regulation:

  • Identify transcription factors regulating ADAMTS19 expression

  • Investigate how methylation affects transcription factor binding

  • Explore the role of non-coding RNAs in ADAMTS19 regulation

How can we develop improved experimental models to study ADAMTS19 function in cancer?

Advancing ADAMTS19 research requires development of sophisticated experimental models:

• Advanced cell culture systems:

  • 3D organoid cultures derived from patient samples with varying ADAMTS19 expression

  • Co-culture systems to study ADAMTS19's role in tumor-stroma interactions

  • Microfluidic devices to assess ADAMTS19's impact on cell migration in real-time

• Genetic manipulation approaches:

  • CRISPR/Cas9-mediated knockout and knock-in models in relevant cell lines

  • Inducible expression systems to study dose-dependent effects

  • Domain-specific mutations to dissect functional roles of different ADAMTS19 regions

• In vivo models:

  • Genetically engineered mouse models with conditional ADAMTS19 expression

  • Patient-derived xenografts to study ADAMTS19 in a more physiological context

  • Orthotopic models specifically for gastric cancer

• High-throughput screening:

  • Develop assays to identify compounds that modulate ADAMTS19 expression or activity

  • Screen for synthetic lethal interactions with ADAMTS19 deficiency

  • Explore therapeutic potential of restoring ADAMTS19 expression in cancers with low expression

What are the key considerations when choosing an ADAMTS19 antibody for specific research applications?

When selecting an ADAMTS19 antibody, consider:

• Target region specificity:

  • For total ADAMTS19 detection: choose antibodies targeting conserved internal regions

  • For specific domain analysis: select antibodies targeting relevant domains

  • Available options include antibodies against internal regions, N-terminal regions, and specific amino acid sequences (AA 325-686, AA 298-328)

• Experimental application:

  • For WB and ELISA: polyclonal antibodies typically provide good sensitivity

  • For IHC/ICC: antibody ab190073 (Abcam) has been validated at 1:1000 dilution

  • For co-IP: antibody ab190073 has demonstrated efficacy

• Species reactivity:

  • Human and mouse reactivity is confirmed for many ADAMTS19 antibodies

  • Predicted cross-reactivity with pig, bovine, horse, sheep, rabbit, and dog exists for some antibodies

• Validation evidence:

  • Prioritize antibodies with published validation in your specific application

  • Consider antibodies used in peer-reviewed publications (like those referenced in the search results)

  • Review manufacturer's validation data for specificity and sensitivity

How should researchers interpret contradictory findings regarding ADAMTS19 expression and function?

When faced with conflicting results:

• Methodological differences:

  • Compare antibodies used (different epitopes may give different results)

  • Evaluate tissue preparation methods (fixation can affect antigen retrieval)

  • Assess quantification methods and cutoff values used for categorization

• Biological context:

  • Consider tissue/tumor heterogeneity and sampling differences

  • Evaluate differences in patient cohorts (ethnicity, treatment history)

  • Assess cancer subtypes/molecular classifications used in different studies

• Functional complexity:

  • ADAMTS19 may have context-dependent functions in different cancer types

  • Protein interactions (like with S100A16) may vary across tissues

  • Post-translational modifications may affect function in ways not detected by expression analysis alone

• Resolution strategies:

  • Perform parallel analyses using multiple methodologies (IHC, WB, qRT-PCR)

  • Include appropriate controls in all experiments

  • Consider meta-analysis of existing data to identify consistent patterns