ADAMTS4 Antibody

What is ADAMTS4 and why is it important in research?

ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4), also known as aggrecanase-1, belongs to the ADAMTS family of extracellular metalloproteinases. This enzyme plays crucial roles in matrix degradation, blood coagulation, and angiogenesis. The ADAMTS family comprises 19 enzymes and 7 ADAMTS-like proteins, with ADAMTS4 functioning as a well-characterized proteoglycanase . It is particularly significant in research because it cleaves aggrecan, a major cartilage proteoglycan, at the '392-Glu-|-Ala-393' site and may be involved in its turnover . ADAMTS4 has been implicated in the pathogenesis of arthritic diseases through its role in cartilage degradation . Additionally, emerging research suggests it could be a critical factor in the exacerbation of neurodegeneration in Alzheimer's disease .

What applications are ADAMTS4 antibodies suitable for?

ADAMTS4 antibodies can be utilized in multiple experimental applications, including:

When selecting an application, researchers should consider that some antibodies may be optimized for specific techniques, and validation in your experimental system is recommended .

How should ADAMTS4 antibodies be stored and handled?

ADAMTS4 antibodies should typically be stored at -20°C for optimal longevity. Most commercial preparations remain stable for at least one year after shipment if properly stored . The antibodies are commonly provided in buffers containing stabilizing agents:

• PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• 0.01M TBS (pH 7.4) with 1% BSA, 0.02% Proclin300, and 50% Glycerol

For antibodies in smaller volumes (e.g., 20µl), preparations may contain 0.1% BSA as a stabilizer . Aliquoting is generally unnecessary for -20°C storage, but repeated freeze-thaw cycles should be avoided to maintain antibody integrity . When handling these antibodies, researchers should be aware that some preparations contain sodium azide, which is toxic and should be disposed of according to appropriate laboratory safety protocols.

What are the key considerations for antibody validation before ADAMTS4 research?

Proper validation of ADAMTS4 antibodies is essential before conducting significant research. Consider these methodological steps:

• Specificity testing: Verify lack of cross-reactivity with other ADAMTS family members. Some antibodies, like PA1-1749A, are specifically designed to avoid cross-reactivity with other ADAMTS proteins , while others may have potential cross-reactivity (e.g., bs-4191R shows potential cross-reactivity with ADAMTS1 due to 73% sequence similarity in the epitope region) .

• Positive control selection: Use appropriate positive controls, such as:

  • Mouse brain tissue for Western blot applications

  • Human liver tissue or human colon cancer tissue for IHC applications

  • Middle-sized intralobular artery in kidney biopsy samples has been validated as a positive control for ADAMTS-4 IHC

• Antibody titration: Determine optimal working concentrations by testing multiple dilutions in your specific experimental system rather than relying solely on manufacturer recommendations .

• Knockout/knockdown validation: When possible, include negative controls using ADAMTS4 knockout or knockdown samples to confirm antibody specificity.

• Multiple antibody comparison: Consider using antibodies that recognize different epitopes of ADAMTS4 to validate your findings.

How should immunohistochemistry protocols be optimized for ADAMTS4 detection in tissue samples?

For optimal ADAMTS4 detection in tissue sections, researchers should consider the following methodological approach:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded (FFPE) tissue sections cut to 4 μm thickness

  • Place sections on charged slides and dry at 60°C for one hour

  • Cool to room temperature before deparaffinizing with appropriate clearing agents

• Antigen retrieval:

  • For optimal results with many ADAMTS4 antibodies, use TE buffer (pH 9.0)

  • Alternatively, citrate buffer (pH 6.0) may be used

  • Heat treatment (e.g., 97°C for 30 minutes) is typically required for effective epitope retrieval

• Antibody incubation:

  • Primary antibody dilutions typically range from 1:50-1:500 for IHC applications

  • Incubate with primary antibody for approximately 60 minutes at room temperature

  • Use appropriate HRP-conjugated secondary antibodies (e.g., incubate for 20 minutes)

• Signal detection and analysis:

  • Develop with 3,3′-diaminobenzidine (DAB) chromogen for approximately 5 minutes

  • Counterstain with hematoxylin for contrast

  • When analyzing results, examine multiple tissue compartments as ADAMTS4 expression may vary by location (e.g., in kidney tissue, evaluate interstitial, glomerular, and tubular compartments)

• Controls and thresholds:

  • Include positive and negative controls in each IHC run

  • Define positive staining threshold (e.g., >1% of observed area showing staining)

  • Consider having multiple independent observers evaluate staining to reduce subjective bias

What are the best practices for detecting various proteolytic fragments of ADAMTS4 in Western blot analysis?

ADAMTS4 undergoes extensive post-translational processing, resulting in multiple proteolytic fragments that can be detected by Western blot. To comprehensively analyze these fragments:

• Sample preparation:

  • Use appropriate extraction buffers containing protease inhibitors to prevent artifactual degradation

  • Consider native versus denaturing conditions depending on your research question

  • Include positive controls (e.g., mouse brain tissue)

• Gel selection and separation:

  • Use gradient gels (e.g., 4-12%) to effectively separate proteins across the ~30-90 kDa range

  • Longer running times may be necessary to achieve optimal separation of closely sized fragments

• Target identification:

  • Primary ADAMTS4 bands typically appear at approximately 68 kDa, 53 kDa, and 30 kDa, representing the full-length protein and its major proteolytic fragments

  • Depending on the extent of C-terminal processing, several smaller bands may also be visible

  • Consider using antibodies that recognize different epitopes to capture all relevant fragments

• Complementary approaches:

  • Some researchers combine multiple ADAMTS4 antibodies (e.g., PA1-1749A with PA1-1750) to detect all processed forms

  • Confirm fragment identity using mass spectrometry or N-terminal sequencing when possible

• Optimization:

  • For Western blot applications, dilutions typically range from 1:300-1:600

  • Longer exposure times may be necessary to visualize less abundant fragments

  • Quantification should focus on all relevant bands to capture total ADAMTS4 expression

How can ADAMTS4 antibodies be utilized to study osteoarthritis progression and potential therapies?

ADAMTS4 (aggrecanase-1) plays a significant role in cartilage degradation during osteoarthritis, making it an important research target for disease progression monitoring and therapeutic development. Advanced methodological approaches include:

• Characterizing ADAMTS4 activity in disease models:

  • Use specific antibodies to monitor ADAMTS4 expression levels in articular cartilage samples from osteoarthritis patients versus controls

  • Correlate ADAMTS4 expression with disease severity and clinical parameters

  • Examine co-localization with aggrecan fragments bearing the NITEGE neoepitope (indicating ADAMTS-mediated cleavage)

• Therapeutic neutralization studies:

  • Evaluate neutralizing antibodies that target both ADAMTS4 and its close relative ADAMTS5 (aggrecanase-2)

  • Screen antibodies for their ability to inhibit aggrecanase activity without cross-reacting with other metalloproteinases of the ADAMTS, ADAM, and MMP families

  • Assess the capacity of candidate antibodies to suppress aggrecanase activity in interleukin-1-stimulated osteoarthritic chondrocytes

• Mechanistic investigations:

  • Use domain-specific antibodies to determine which structural domains are essential for ADAMTS4 activity in cartilage

  • Investigate post-translational modifications and their impact on ADAMTS4 function

  • Examine the interaction between ADAMTS4 and tissue inhibitors or activators

• Translational approaches:

  • Develop and validate biomarker assays using ADAMTS4 antibodies to monitor treatment response

  • Investigate the relationship between ADAMTS4 levels in synovial fluid or circulation and disease progression

  • Explore dual inhibition of ADAMTS4 and ADAMTS5 as a potential therapeutic strategy for osteoarthritis

What methodologies are recommended for investigating ADAMTS4's role in neurological disorders?

Recent research suggests ADAMTS4 may play a role in neurological conditions, particularly Alzheimer's disease . Advanced methodological approaches for investigating this connection include:

• Tissue-specific expression analysis:

  • Utilize immunohistochemistry to map ADAMTS4 expression patterns in different brain regions, with particular attention to areas affected in neurodegeneration

  • Compare ADAMTS4 levels in neural tissues from patients with Alzheimer's disease versus age-matched controls

  • Examine co-localization with amyloid plaques, neurofibrillary tangles, and inflammatory markers

• Functional studies in neural models:

  • Employ ADAMTS4 antibodies in primary neural cell cultures or brain organoids to investigate the effects of ADAMTS4 inhibition on proteoglycan turnover

  • Use neutralizing antibodies to block ADAMTS4 function in relevant experimental models of neurodegeneration

  • Investigate changes in synaptic function and neuronal survival in response to ADAMTS4 modulation

• Substrate identification:

  • Use co-immunoprecipitation with ADAMTS4 antibodies followed by mass spectrometry to identify novel neural substrates

  • Validate substrate cleavage using in vitro assays and generate neoepitope antibodies to detect specific cleavage products in vivo

  • Investigate the relationship between ADAMTS4 activity and the metabolism of key proteins implicated in neurodegeneration

• Mechanistic pathways:

  • Study the regulation of ADAMTS4 expression in response to inflammatory mediators relevant to neurological disorders

  • Investigate potential interactions between ADAMTS4 and components of the blood-brain barrier

  • Examine how ADAMTS4-mediated extracellular matrix remodeling might influence neural plasticity and regeneration

How can researchers effectively distinguish between ADAMTS4 and closely related family members in complex biological samples?

The ADAMTS family comprises 19 enzymes with structural similarities, creating challenges for specific detection . Advanced approaches for ensuring ADAMTS4 specificity include:

• Epitope selection and antibody screening:

  • Choose antibodies raised against unique regions of ADAMTS4 with minimal sequence homology to other family members

  • Be aware of potential cross-reactivity issues, such as between ADAMTS4 and ADAMTS1 (which may share 73% sequence similarity in some epitope regions)

  • Validate specificity using recombinant proteins for multiple ADAMTS family members

• Combined antibody approaches:

  • Utilize antibodies targeting different domains of ADAMTS4 to increase confidence in identification

  • Consider using a combination of monoclonal and polyclonal antibodies for confirmation

  • Some commercial antibodies, like PA1-1749A, have been specifically validated for lack of cross-reactivity with other ADAMTS family members

• Activity-based discrimination:

  • Develop functional assays that distinguish ADAMTS4 from related enzymes based on substrate specificity

  • Combine with neutralizing antibodies to confirm the contribution of ADAMTS4 versus other family members

  • Incorporate specific inhibitors alongside antibody detection to validate results

• Molecular approaches:

  • Use genetic knockdown or knockout systems to validate antibody specificity

  • Consider advanced techniques like proximity ligation assays to confirm specific ADAMTS4 detection

  • Employ mass spectrometry following immunoprecipitation to unambiguously identify ADAMTS4 and distinguish it from related proteins

How should researchers address inconsistent ADAMTS4 detection in different tissue types?

ADAMTS4 expression and detectability can vary considerably across tissues, potentially leading to inconsistent results. Methodological approaches to address this challenge include:

• Tissue-specific optimization:

  • Adjust fixation protocols based on tissue type (different tissues may require modified fixation times or conditions)

  • Optimize antigen retrieval methods for specific tissues (e.g., TE buffer pH 9.0 versus citrate buffer pH 6.0)

  • Consider tissue-specific positive controls (e.g., mouse brain tissue for Western blot, human liver or colon cancer tissue for IHC)

• Antibody selection considerations:

  • Different antibodies may perform optimally in specific tissues

  • For challenging tissues, consider testing multiple antibodies targeting different ADAMTS4 epitopes

  • Be aware that some tissues may express different ADAMTS4 isoforms or processed forms that affect antibody recognition

• Signal amplification strategies:

  • For tissues with low ADAMTS4 expression, implement signal amplification systems such as tyramide signal amplification

  • Consider more sensitive detection systems for Western blotting (e.g., enhanced chemiluminescence substrates)

  • Employ primary antibody signal amplifiers like Rabbit Linker systems for IHC applications

• Compartmentalized analysis approach:

  • Analyze ADAMTS4 expression separately in different tissue compartments

  • In kidney tissue, for example, examine interstitial (peritubular capillaries, interstitial stroma), glomerular (glomerular capillaries, Bowman space), and tubular (proximal and distal) compartments independently

  • Define clear positivity thresholds (e.g., >1% of observed area showing staining)

What strategies can improve detection of low-abundance ADAMTS4 in experimental samples?

ADAMTS4 may be present at low levels in certain tissues or experimental conditions, presenting detection challenges. Consider these methodological approaches:

• Sample preparation enhancements:

  • Implement protein concentration techniques (e.g., immunoprecipitation) before Western blot analysis

  • Use protease inhibitor cocktails to prevent degradation during sample processing

  • Consider subcellular fractionation to enrich for secreted proteins including ADAMTS4

• Optimized antibody protocols:

  • Increase primary antibody concentration while carefully monitoring background

  • Extend primary antibody incubation time (e.g., overnight at 4°C)

  • Utilize higher sensitivity detection systems (e.g., SuperSignal™ or similar enhanced chemiluminescent substrates)

• Signal amplification methods:

  • Employ biotin-streptavidin amplification systems where appropriate

  • Consider tyramide signal amplification for IHC applications

  • Use primary antibody signal amplifiers (such as Rabbit Linker Dako GV809) before secondary antibody application

• Alternative detection platforms:

  • For quantitative analysis of low-abundance ADAMTS4, consider using ultrasensitive ELISA techniques

  • Explore digital ELISA platforms or other single-molecule detection methods

  • Consider mass spectrometry-based approaches for detection and quantification

How can researchers effectively interpret ADAMTS4 antibody results that show multiple bands in Western blot analysis?

ADAMTS4 undergoes extensive proteolytic processing, resulting in multiple protein bands during Western blot analysis. To interpret these complex patterns correctly:

• Understanding the expected pattern:

  • ADAMTS4 typically appears as multiple bands around 68 kDa, 53 kDa, and 30 kDa, representing the full-length protein and its proteolytic fragments

  • Additional smaller bands may appear depending on the extent of C-terminal processing

  • The pattern of bands can provide information about post-translational processing in your experimental system

• Distinguishing specific from non-specific binding:

  • Compare observed bands with the calculated molecular weight (90 kDa) and commonly observed weights

  • Include appropriate positive controls (e.g., mouse brain tissue)

  • Consider using multiple antibodies targeting different epitopes to confirm band identity

• Advanced validation approaches:

  • Confirm band identity through immunoprecipitation followed by mass spectrometry

  • Use ADAMTS4 knockout/knockdown controls to identify which bands disappear

  • Consider treating samples with recombinant ADAMTS4 to generate a comparison pattern of proteolytic fragments

• Quantification considerations:

  • When quantifying ADAMTS4 levels, determine whether to measure a specific band or sum multiple bands

  • Be consistent in your quantification approach across experimental conditions

  • Consider the biological significance of shifts in the ratio between different bands (which may indicate altered processing)

What is the potential for using ADAMTS4 antibodies in developing diagnostic or prognostic biomarkers?

Recent research points to ADAMTS4 as a potential biomarker in several pathological conditions, including osteoarthritis and neurological disorders. Methodological considerations for biomarker development include:

• Biological fluid analysis:

  • Investigate ADAMTS4 levels in accessible biofluids (serum, plasma, synovial fluid, cerebrospinal fluid)

  • Correlate ADAMTS4 levels with disease severity and progression

  • Develop standardized ELISA protocols using validated ADAMTS4 antibodies for reliable quantification

• Tissue-based diagnostic approaches:

  • Establish standardized immunohistochemistry scoring systems for ADAMTS4 in disease-relevant tissues

  • Evaluate whether ADAMTS4 expression patterns correlate with clinical outcomes

  • Consider combining ADAMTS4 with other markers for improved diagnostic accuracy

• Target validation strategies:

  • Confirm the relationship between ADAMTS4 levels and disease mechanisms through interventional studies

  • Investigate whether changes in ADAMTS4 levels precede clinical manifestations, suggesting prognostic value

  • Determine whether ADAMTS4 levels respond to therapeutic interventions, indicating potential as a treatment response marker

• Technical assay development:

  • Optimize antibody pairs for sandwich ELISA development

  • Validate assay performance characteristics (sensitivity, specificity, reproducibility)

  • Consider automated platforms for high-throughput clinical applications

How can ADAMTS4 antibodies be utilized in exploring cross-talk between inflammatory pathways and extracellular matrix remodeling?

ADAMTS4 operates at the intersection of inflammation and matrix remodeling. Advanced research strategies include:

• Co-localization studies:

  • Use dual immunostaining to examine spatial relationships between ADAMTS4 and inflammatory mediators

  • Investigate co-expression with cytokines, chemokines, and their receptors in disease tissues

  • Analyze the relationship between ADAMTS4 expression and inflammatory cell infiltration

• Functional interaction analysis:

  • Employ ADAMTS4 antibodies in combination with neutralizing antibodies against key inflammatory mediators

  • Investigate how modulating inflammatory pathways affects ADAMTS4 expression and activity

  • Examine feedback mechanisms whereby ADAMTS4-mediated matrix degradation influences inflammatory responses

• Signaling pathway investigations:

  • Use phospho-specific antibodies alongside ADAMTS4 detection to map activated signaling pathways

  • Determine how inflammatory stimuli regulate ADAMTS4 transcription, translation, and post-translational modifications

  • Investigate whether ADAMTS4-generated matrix fragments act as damage-associated molecular patterns (DAMPs) that propagate inflammation

• Therapeutic implications:

  • Explore the potential of dual targeting strategies addressing both inflammation and ADAMTS4 activity

  • Assess whether ADAMTS4 inhibition modifies inflammatory responses in preclinical models

  • Investigate temporal relationships between inflammatory events and ADAMTS4 upregulation to identify optimal intervention windows

How should researchers reconcile conflicting results when different ADAMTS4 antibodies yield varying patterns of expression?

When different ADAMTS4 antibodies produce inconsistent results, employ these methodological approaches:

• Epitope mapping analysis:

  • Compare the epitopes recognized by different antibodies to understand potential reasons for discrepancies

  • Consider whether antibodies target domains that may be lost during proteolytic processing

  • Evaluate whether post-translational modifications might affect epitope accessibility

• Validation through orthogonal techniques:

  • Confirm antibody findings using non-antibody-based methods (e.g., mRNA expression analysis)

  • Consider activity-based assays to confirm functional ADAMTS4 presence

  • Implement genetic approaches (siRNA knockdown, CRISPR knockout) to validate antibody specificity

• Technical factor assessment:

  • Systematically evaluate whether methodological differences (fixation, antigen retrieval, detection systems) account for discrepancies

  • Test antibodies side-by-side under identical conditions

  • Investigate whether differences relate to antibody format (monoclonal vs. polyclonal) or host species

• Biological interpretation:

  • Consider whether discrepancies reflect detection of different ADAMTS4 isoforms or processed forms

  • Evaluate tissue-specific or context-dependent post-translational modifications

  • Assess whether conflicting results might actually reveal novel aspects of ADAMTS4 biology

What considerations are important when quantifying ADAMTS4 expression levels across experimental conditions?

Accurate quantification of ADAMTS4 requires careful methodological attention:

• Standardization approaches:

  • Develop consistent protocols for sample collection, processing, and analysis

  • Include calibration standards and quality controls in each experimental run

  • Normalize ADAMTS4 measurements to appropriate housekeeping proteins or reference genes

• Dynamic range considerations:

  • Ensure detection methods can accurately measure across the full range of expected ADAMTS4 expression

  • When using immunohistochemistry, establish clear scoring criteria (e.g., positive staining defined as >1% of observed area)

  • For quantitative Western blot, ensure linearity of signal across the concentration range of interest

• Multi-compartment analysis:

  • Consider analyzing ADAMTS4 expression separately in different cellular compartments

  • In kidney tissue, for example, evaluate interstitial, glomerular, and tubular compartments independently

  • Be aware that ADAMTS4 distribution may vary between intracellular, cell-surface, and extracellular locations

• Statistical approaches:

  • Apply appropriate statistical methods based on data distribution

  • Consider using multiple independent observers to reduce subjective bias in scoring

  • Be transparent about variability and potential confounding factors

What novel approaches might enhance the specificity and sensitivity of ADAMTS4 detection in complex biological systems?

Emerging technologies offer promising avenues for improved ADAMTS4 detection:

• Advanced antibody engineering:

  • Develop recombinant antibodies with enhanced specificity for ADAMTS4

  • Create conformation-specific antibodies that distinguish active from inactive ADAMTS4

  • Engineer bispecific antibodies that simultaneously recognize two distinct ADAMTS4 epitopes for improved specificity

• Activity-based probes:

  • Design chemical probes that covalently bind to the active site of ADAMTS4

  • Develop FRET-based biosensors that report on ADAMTS4 enzymatic activity in real-time

  • Create activity-based nanoparticles for in vivo imaging of ADAMTS4 function

• Single-cell technologies:

  • Apply mass cytometry (CyTOF) with metal-conjugated ADAMTS4 antibodies for high-dimensional analysis

  • Develop spatial transcriptomics approaches that correlate ADAMTS4 protein expression with its transcriptional landscape

  • Implement imaging mass spectrometry to map ADAMTS4 distribution with subcellular resolution

• Computational approaches:

  • Apply machine learning algorithms to analyze complex ADAMTS4 expression patterns

  • Develop predictive models that integrate multiple data types (transcriptomic, proteomic, clinical)

  • Create network analysis tools to understand ADAMTS4 in the context of broader biological systems

How might ADAMTS4 antibodies contribute to understanding tissue-specific differences in enzyme function across disease states?

ADAMTS4 may play distinct roles in different tissues and pathological conditions. Advanced research strategies include:

• Comparative tissue analysis:

  • Systematically compare ADAMTS4 expression patterns across multiple tissue types in health and disease

  • Investigate tissue-specific post-translational modifications that might alter ADAMTS4 function

  • Examine whether ADAMTS4 associates with different binding partners in various tissue microenvironments

• Disease progression mapping:

  • Track changes in ADAMTS4 expression, localization, and processing across disease stages

  • Correlate ADAMTS4 patterns with tissue-specific pathological changes

  • Investigate whether ADAMTS4 plays different roles at initiation versus progression phases of disease

• Substrate landscape exploration:

  • Use proteomics approaches to identify tissue-specific ADAMTS4 substrates

  • Develop neoepitope antibodies that detect specific ADAMTS4 cleavage products in different tissues

  • Compare the efficiency of ADAMTS4-mediated proteolysis across tissue types

• Regulatory mechanism investigation:

  • Examine tissue-specific transcriptional and post-transcriptional regulation of ADAMTS4

  • Investigate whether environmental factors differently modulate ADAMTS4 in various tissues

  • Explore epigenetic mechanisms that might contribute to tissue-specific ADAMTS4 expression patterns