ADSS Antibody

What is ADSS and why is it significant in biomedical research?

ADSS (Adenylosuccinate synthetase) is a key enzyme involved in the purine biosynthetic pathway, playing a crucial role in the production of purine nucleotides essential for DNA and RNA synthesis. It exists as multiple isozymes, with ADSS2 (also known as adenylosuccinate synthetase 2) being the predominant form studied in research contexts. The enzyme is localized in both mitochondria and cytoplasm and is widely expressed across various tissue types . Its significance stems from its fundamental role in cellular metabolism and its dysregulation in various diseases, particularly cancer, making it an attractive target for therapeutic interventions and biomarker research .

What are the key characteristics of ADSS that researchers should understand?

ADSS has a canonical amino acid length of 456 residues and a protein mass of approximately 50.1 kilodaltons in humans . The ADSS gene sequence spans 44 kb, containing 13 exons and 12 introns . Researchers should be aware that ADSS might also be referred to by synonyms such as ADSS2, AMPSase 2, IMP--aspartate ligase 2, or L-type adenylosuccinate synthetase in scientific literature . The enzyme shows moderate cytoplasmic immunoreactivity in malignant cells, with notably high expression in certain cases of malignant lymphoma, breast cancer, and colorectal cancer . Understanding these characteristics is essential for proper experimental design and interpretation of results when working with ADSS antibodies.

What types of ADSS antibodies are available for research, and how should researchers select the appropriate one?

Researchers have access to several types of ADSS antibodies, with the most common being rabbit polyclonal antibodies. These antibodies typically recognize the endogenous levels of total ADSS protein . When selecting an ADSS antibody, researchers should consider:

• Application compatibility: Verify that the antibody has been validated for your specific application (e.g., Western blot, immunofluorescence, ELISA)

• Species reactivity: Ensure the antibody recognizes ADSS in your experimental model organism (human, mouse, rat, etc.)

• Specificity: Check for cross-reactivity with other proteins or ADSS isoforms

• Immunogen information: Consider whether the antibody was raised against full-length protein or a specific epitope

• Validation data: Examine available scientific validation data showing the antibody's performance in relevant applications

Selection should be based on the specific research questions being addressed and the experimental techniques planned.

What are the optimal protocols for using ADSS antibodies in Western blot applications?

For effective Western blot detection of ADSS using antibodies, researchers should follow this methodological approach:

• Sample preparation: Extract proteins from cells or tissues using appropriate lysis buffers that preserve ADSS structure while minimizing degradation.

• Gel electrophoresis and transfer: Separate proteins using SDS-PAGE (typically 10-12% gels are suitable for the ~50kDa ADSS protein) and transfer to PVDF or nitrocellulose membranes.

• Blocking: Block membranes with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute anti-ADSS antibody (typically 1:1000 to 1:2000) in blocking buffer and incubate overnight at 4°C. The exact dilution may vary by antibody source and should be optimized for each application .

• Washing: Wash 3-5 times with TBST, 5 minutes each wash.

• Secondary antibody incubation: Incubate with HRP-conjugated secondary antibody (anti-rabbit IgG for most ADSS antibodies) at an appropriate dilution (typically 1:5000 to 1:10000) for 1 hour at room temperature .

• Detection: Develop using chemiluminescent substrate and image using an appropriate detection system.

The expected result should show a band at approximately 50.1 kDa corresponding to ADSS protein, as evidenced by validation data showing successful detection in various cell lines .

How can researchers optimize immunofluorescence protocols for ADSS localization studies?

For accurate ADSS localization using immunofluorescence, researchers should follow this optimized protocol:

• Cell preparation: Culture cells on coverslips or chamber slides at appropriate density (70-80% confluence is optimal).

• Fixation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature. Alternative fixatives like methanol may be used depending on the epitope accessibility.

• Permeabilization: Permeabilize cells with 0.1-0.5% Triton X-100 in PBS for 10 minutes.

• Blocking: Block with 5% normal serum (from the same species as the secondary antibody) in PBS with 0.1% Triton X-100 for 1 hour.

• Primary antibody incubation: Apply diluted ADSS antibody (typically 1:50 to 1:200 in blocking solution) and incubate overnight at 4°C in a humidified chamber .

• Washing: Wash 3 times with PBS, 5 minutes each.

• Secondary antibody incubation: Incubate with fluorophore-conjugated secondary antibody (e.g., FITC-conjugated anti-rabbit IgG) at 1:200-1:500 dilution for 1 hour at room temperature in the dark .

• Counterstaining: Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes.

• Mounting: Mount with anti-fade mounting medium.

Researchers should expect to observe ADSS primarily in the cytoplasm with some mitochondrial localization, as shown in validation images of HeLa cells where ADSS displays cytoplasmic distribution with DAPI highlighting the nuclei .

What methodological approaches should be used to validate ADSS antibody specificity?

Validating ADSS antibody specificity requires a multi-pronged approach to ensure reliable experimental results:

• Genetic knockdown/knockout validation: Use siRNA or CRISPR/Cas9 to reduce or eliminate ADSS expression, then confirm reduced signal in antibody-based assays. For example, lentiviral vector-mediated ADSS knockdown using specifically designed siRNAs (such as hADSS-1304-s: AGCUCAAAUUCCAGUUAA or HADSS-1304-a: UUAACUGAAUUUGAAGCU) can be used to validate antibody specificity .

• Overexpression studies: Transiently overexpress tagged ADSS in cells and confirm co-localization of tag and antibody signals.

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide or recombinant ADSS protein (which was used as the immunogen for many commercial antibodies) before application to samples; specific signal should be significantly reduced .

• Cross-species reactivity testing: Test the antibody on samples from multiple species if the epitope is conserved, confirming signal at the appropriate molecular weight.

• Mass spectrometry validation: Perform immunoprecipitation with the ADSS antibody followed by mass spectrometry to confirm the identity of the captured protein.

• Multiple antibody validation: Compare results using antibodies raised against different epitopes of ADSS to confirm consistent patterns.

These validation methods help ensure that observed signals truly represent ADSS protein rather than non-specific binding or cross-reactivity.

How can ADSS antibodies be utilized in cancer research, particularly for breast cancer studies?

ADSS antibodies offer valuable tools for investigating ADSS's role in cancer, with particular relevance to breast cancer research:

• Expression profiling: ADSS antibodies can be used in immunohistochemistry, Western blotting, or tissue microarrays to analyze ADSS expression patterns across breast cancer subtypes, stages, and in comparison to normal tissue. Research has shown that ADSS is highly expressed in breast cancer and its subtypes, making it a potential marker for malignancy .

• Prognostic biomarker validation: Recent studies have identified ADSS as associated with poor prognosis in breast cancer. Researchers can use ADSS antibodies to correlate expression levels with clinical outcomes, including survival data and treatment response .

• ADSS-based scoring models: Antibody-based detection of ADSS can be incorporated into clinical scoring models. For example, a recently developed ADSS-related scoring model showed significant prognostic impact on clinical characteristics such as metastasis to lymph nodes .

• Immunomodulatory studies: ADSS antibodies can be used to investigate how ADSS levels affect the immune microenvironment in breast cancer tissues, as research suggests ADSS scores and related genes may influence immune responses in breast cancer patients .

• Therapeutic target identification: Using ADSS antibodies, researchers can evaluate the effects of ADSS inhibition on cancer cell proliferation and survival, potentially leading to new therapeutic approaches.

This multi-faceted approach using ADSS antibodies helps elucidate ADSS's role in breast cancer pathogenesis and its potential as a therapeutic target.

What experimental designs are recommended for studying ADSS inhibition as a potential cancer therapeutic approach?

When investigating ADSS inhibition as a cancer therapeutic strategy, researchers should consider the following experimental design approaches:

• In vitro inhibition studies:

  • Use ADSS antibodies to monitor protein levels before and after treatment with potential inhibitors

  • Design dose-response experiments with siRNA knockdown or small molecule inhibitors

  • Measure cell proliferation, apoptosis, and metabolic changes following ADSS inhibition

  • Include appropriate positive and negative controls in all experiments

• Mechanistic studies:

  • Investigate the effects of ADSS inhibition on purine biosynthesis pathways

  • Examine downstream metabolic changes using metabolomics approaches

  • Use ADSS antibodies in co-immunoprecipitation experiments to identify interaction partners that might be affected by inhibition

• Combination therapy assessment:

  • Test ADSS inhibition in combination with standard chemotherapeutics to identify synergistic effects

  • Use ADSS antibodies to monitor changes in expression or localization during combination treatment

• In vivo validation:

  • Develop xenograft models with varying ADSS expression levels

  • Use ADSS antibodies for immunohistochemical analysis of tumors following treatment

  • Correlate ADSS expression with tumor growth, metastasis, and response to therapy

• Resistance mechanism identification:

  • Apply ADSS antibodies to study adaptive responses to ADSS inhibition

  • Characterize resistant cell populations for changes in ADSS expression or localization

These experimental approaches provide a comprehensive framework for evaluating ADSS as a therapeutic target in cancer, particularly in breast cancer where ADSS has been shown to be highly expressed and associated with poor prognosis .

How do ADSS antibodies contribute to understanding the immune microenvironment in cancer research?

ADSS antibodies provide critical tools for investigating the complex relationship between ADSS expression and the tumor immune microenvironment:

• Multiplex immunofluorescence analysis: ADSS antibodies can be used in multiplex immunofluorescence panels alongside immune cell markers to visualize the spatial relationship between ADSS-expressing cells and immune cell infiltrates. This approach helps identify whether high ADSS expression correlates with specific immune cell populations or their functional states.

• Flow cytometry applications: Using ADSS antibodies in flow cytometry enables researchers to quantify ADSS expression in specific cell populations within the tumor microenvironment and correlate this with immune cell markers.

• Immunosuppressive mechanism investigation: Recent research indicates that the ADSS score may affect the immune microenvironment of breast cancer patients . ADSS antibodies can help elucidate whether high ADSS expression contributes to immunosuppression through metabolic competition for purines or other mechanisms.

• Checkpoint inhibitor response correlation: By examining ADSS expression using antibodies in patient samples, researchers can investigate whether ADSS levels predict response to immune checkpoint inhibitors, potentially identifying a new biomarker for immunotherapy efficacy.

• Therapeutic monitoring: In experimental models testing ADSS-targeting therapies, ADSS antibodies allow researchers to monitor changes in the immune microenvironment during treatment, potentially revealing mechanisms of action or resistance.

This integrated approach helps establish ADSS not only as a metabolic enzyme but potentially as an immunomodulatory factor in cancer, opening new avenues for targeted therapies that may synergize with existing immunotherapies.

What are common challenges in interpreting ADSS antibody data in complex tissue samples, and how can researchers address them?

Researchers face several challenges when interpreting ADSS antibody data in complex tissues:

• Heterogeneous expression patterns:

  • Challenge: ADSS shows variable expression across different cell types within the same tissue, making quantification difficult.

  • Solution: Use single-cell analytical approaches or laser capture microdissection to isolate specific cell populations before antibody-based analysis. Additionally, employ digital image analysis software to quantify expression in specific regions or cell types identified by co-staining with cell-type-specific markers.

• Background and non-specific binding:

  • Challenge: Especially in tissues with high lipid content or autofluorescence, distinguishing true ADSS signal from background can be problematic.

  • Solution: Optimize blocking conditions (5% BSA or specialized blocking reagents), include appropriate negative controls (isotype controls, secondary-only controls), and consider tissue-specific antigen retrieval methods. For immunohistochemistry, DAB color development should be carefully controlled to prevent background staining while allowing visualization of true signal .

• Isoform-specific detection:

  • Challenge: Distinguishing between ADSS isoforms or closely related proteins.

  • Solution: Select antibodies with documented specificity for the isoform of interest, and validate results using complementary methods such as RT-PCR for isoform-specific transcripts.

• Quantification standardization:

  • Challenge: Comparing ADSS expression levels across different experimental batches or studies.

  • Solution: Include standardized positive controls in each experiment, normalize to housekeeping proteins, and consider using automated image analysis with consistent thresholding parameters.

• Epitope masking in disease states:

  • Challenge: Disease-related modifications might mask the ADSS epitope recognized by the antibody.

  • Solution: Use multiple antibodies recognizing different epitopes and correlate results with functional assays or mRNA expression.

By implementing these strategies, researchers can improve the reliability and reproducibility of ADSS antibody-based analyses in complex tissue samples.

How should researchers approach conflicting or unexpected ADSS antibody results?

When faced with conflicting or unexpected ADSS antibody results, researchers should follow this systematic troubleshooting approach:

• Technical validation:

  • Repeat the experiment with fresh reagents and samples

  • Test multiple ADSS antibodies targeting different epitopes

  • Verify the antibody lot and storage conditions, as antibody degradation can cause inconsistent results

  • Confirm protein loading, transfer efficiency, and detection sensitivity in Western blots

• Biological explanation assessment:

  • Consider whether experimental conditions might be affecting ADSS expression or localization

  • Investigate whether post-translational modifications might be masking epitopes or altering antibody binding

  • Explore whether experimental manipulations have affected ADSS at the transcriptional level using qRT-PCR

• Complementary methodologies:

  • Validate antibody-based results using orthogonal techniques:

    • mRNA expression analysis with qRT-PCR or RNA-seq

    • Activity assays to measure ADSS enzymatic function

    • Mass spectrometry to confirm protein identity and abundance

• Reconciliation strategies:

  • When using different antibodies yields conflicting results, determine which antibody has the most extensive validation data

  • Consider creating a knockout or knockdown validation system as described in breast cancer studies using lentiviral vector-mediated ADSS siRNA

  • Analyze subcellular fractions separately, as ADSS localizes to multiple cellular compartments which may explain differential detection

• Result contextualization:

  • Review literature for similar discrepancies and how they were resolved

  • Consider whether experimental models accurately represent in vivo conditions

  • Evaluate whether biological variability rather than technical issues might explain the results

This methodical approach helps distinguish genuine biological insights from technical artifacts when working with ADSS antibodies.

What considerations are important when analyzing ADSS expression data in relation to clinical outcomes?

When correlating ADSS expression data with clinical outcomes, researchers should address these critical considerations:

• Standardized scoring systems:

  • Develop or adopt quantitative scoring methods for ADSS expression that account for both staining intensity and percentage of positive cells

  • Consider using digital pathology platforms for objective quantification

  • Establish clear cutoff values for defining "high" versus "low" expression based on statistical analyses of the cohort

• Patient stratification factors:

  • Account for key clinical variables including:

    • Cancer subtype (particularly important in breast cancer where different molecular subtypes may show varying ADSS dependencies)

    • Stage of disease

    • Prior treatment history

    • Age and comorbidities

  • Perform multivariate analyses to determine if ADSS is an independent prognostic factor

• Temporal considerations:

  • Evaluate whether ADSS expression changes during disease progression

  • Consider analyzing paired samples (pre- and post-treatment) to assess dynamic changes

  • Account for the time point at which samples were collected in relation to treatment

• Microenvironmental context:

  • Analyze ADSS expression in relation to the immune microenvironment, as recent evidence suggests ADSS scores may affect immune cell infiltration in breast cancer

  • Consider using multiplex immunohistochemistry to assess ADSS expression alongside immune cell markers

• Statistical approach:

  • Use appropriate statistical methods for survival analysis (e.g., Kaplan-Meier curves, Cox proportional hazards models)

  • Adjust for multiple hypothesis testing when necessary

  • Consider sample size limitations and potential biases in retrospective analyses

• Validation cohorts:

  • Confirm findings in independent patient cohorts

  • Consider using public datasets (e.g., TCGA, GEO) for external validation of ADSS mRNA expression correlation with outcomes

Following these considerations helps ensure robust and clinically meaningful analysis of ADSS expression in relation to patient outcomes, potentially leading to validated prognostic models as demonstrated in recent breast cancer research .

How can ADSS antibodies be incorporated into anti-drug antibody (ADA) assays for immunogenicity testing?

While ADSS antibodies and anti-drug antibodies (ADAs) represent different research areas, ADSS antibodies can serve as controls or tools in developing robust ADA assays:

• Positive control development:

  • Well-characterized ADSS antibodies with defined specificity and affinity can serve as reference standards when establishing new immunogenicity assay platforms

  • These antibodies provide consistent reactivity benchmarks for assay validation

• Cross-reactivity assessment:

  • ADSS antibodies can help validate that anti-drug antibody assays are specific to the therapeutic drug and don't cross-react with endogenous proteins

  • Include ADSS antibodies in specificity panels during ADA assay development

• Immunogenicity testing workflow integration:

  • In the standard immunogenicity testing schematic that includes screening, confirmation, and titration assays , ADSS antibodies can serve as system suitability controls

  • They provide a way to ensure consistency across different testing occasions

• Data analysis standardization:

  • When converting immunogenicity data from SDTM (Study Data Tabulation Model) to ADaM (Analysis Data Model) structure as recommended by regulatory agencies , include ADSS antibody testing data as reference points

• Multiplexed analysis platforms:

  • In advanced multiplex platforms for simultaneous detection of multiple ADAs, include ADSS antibody detection as an internal control

  • This helps normalize results across different experimental conditions

By incorporating ADSS antibodies into ADA assay development and validation workflows, researchers can enhance the reliability and standardization of immunogenicity testing, which is becoming an increasingly important aspect of therapeutic antibody development .

What novel applications are emerging for ADSS antibodies in native mass spectrometry and ion mobility studies?

The integration of ADSS antibodies with advanced mass spectrometry techniques is opening new research avenues:

• Native MS characterization of ADSS complexes:

  • ADSS antibodies can be used to immunoprecipitate ADSS and its binding partners for subsequent native MS analysis

  • This approach preserves protein-protein interactions, allowing researchers to study ADSS in its biological context

  • Native MS provides accurate mass measurement of intact protein complexes with high resolution (<30 ppm)

• Ion mobility MS (IM-MS) for conformational analysis:

  • ADSS antibodies can help isolate pure ADSS for IM-MS studies

  • IM-MS can measure collisional cross sections of ADSS under different conditions, revealing conformational changes associated with disease states or drug binding

  • This technique is particularly valuable for detecting subtle structural alterations that might not be apparent with other methods

• Antibody-drug conjugate (ADC) research applications:

  • ADSS antibodies could potentially be developed into ADCs for targeted therapy

  • Native MS and IM-MS are powerful tools for characterizing ADC quality attributes including drug distribution, amount of naked antibody, and average drug-to-antibody ratio (DAR)

  • These methods can provide insights into the structural heterogeneity of potential ADSS-targeting therapeutic antibodies

• Hybrid structural biology approaches:

  • Combining ADSS antibody epitope mapping with native MS and IM-MS creates a comprehensive structural biology workflow

  • This integrated approach can reveal how ADSS structure relates to its function in normal and disease states

  • The semiquantitative interpretation of IM-MS data allows researchers to directly extrapolate structural characteristics of antibody-ADSS complexes

These emerging applications represent the cutting edge of antibody-based structural biology, offering unprecedented insights into ADSS structure, function, and potential therapeutic targeting.

What methodological approaches are recommended for developing and validating ADSS inhibitors using antibody-based assays?

For researchers developing ADSS inhibitors, antibody-based assays provide critical tools for compound validation and mechanism studies:

• Target engagement verification:

  • Use cellular thermal shift assays (CETSA) with ADSS antibodies to confirm direct binding of inhibitors to ADSS in cells

  • This technique measures the thermal stabilization of ADSS upon inhibitor binding, providing evidence of direct engagement

  • Western blot with ADSS antibodies is used as the readout for protein levels after thermal challenge

• Structure-activity relationship (SAR) studies:

  • Develop competitive binding assays where candidate inhibitors compete with ADSS antibodies for binding to the enzyme

  • The degree of competition provides insights into binding site overlap and relative affinity

  • These assays can be formatted as ELISA or AlphaScreen platforms for higher throughput

• Phenotypic assay development:

  • Create cell-based assays where ADSS inhibition produces measurable phenotypes

  • Use ADSS antibodies to correlate inhibitor effects with ADSS levels or localization changes

  • Develop immunofluorescence-based high-content screening assays using optimized ADSS antibody protocols

• Resistance mechanism characterization:

  • When resistance to ADSS inhibitors develops, use ADSS antibodies to investigate:

    • Changes in expression levels

    • Post-translational modifications

    • Subcellular relocalization

    • Altered complex formation with binding partners

• Biomarker development:

  • Establish ADSS antibody-based assays as pharmacodynamic biomarkers

  • Quantify changes in downstream pathways using phospho-specific antibodies to related signaling molecules

  • Develop multiplex panels including ADSS and other relevant targets

• Combination therapy rational design:

  • Use ADSS antibodies to monitor changes in ADSS expression or localization following treatment with other agents

  • Identify synergistic combinations based on mechanistic insights from these studies

These methodological approaches create a comprehensive framework for ADSS inhibitor development, guided by antibody-based assays that provide crucial molecular insights throughout the drug discovery process.