ASS1 Recombinant Monoclonal Antibody

What is ASS1 and what is its biological function in mammalian systems?

ASS1 (Argininosuccinate Synthase 1) is a ubiquitous enzyme that catalyzes the formation of argininosuccinate from citrulline and aspartate using ATP. It functions as a critical component of the urea cycle, which processes excess nitrogen generated during protein catabolism to produce urea, which is subsequently excreted in urine . ASS1 catalyzes the penultimate step in the arginine biosynthetic pathway and is primarily expressed in periportal hepatocytes but also found in most other body tissues . The protein has a tetrameric structure composed of identical subunits and has a molecular weight of approximately 47 kDa .

ASS1 deficiency causes citrullinemia (CTLN1), an autosomal recessive urea cycle disorder characterized by elevated plasma citrulline levels, hyperammonemia, vomiting spells, and potential mental retardation . Recent research has also identified ASS1 as a potential tumor suppressor in various cancers, including breast cancer .

What applications are suitable for ASS1 recombinant monoclonal antibodies?

ASS1 recombinant monoclonal antibodies are validated for multiple research applications:

The optimal working dilution should be determined empirically by each researcher based on their specific experimental conditions .

What species reactivity is typically available for ASS1 recombinant monoclonal antibodies?

ASS1 antibodies exhibit varying species reactivity profiles depending on the clone and manufacturer:

This multi-species reactivity is particularly valuable for comparative studies across model organisms .

How should ASS1 recombinant monoclonal antibodies be stored to maintain optimal activity?

For optimal preservation of ASS1 antibody activity:

• Short-term storage (up to 1 month): 4°C

• Long-term storage: -20°C

• Avoid repeated freeze-thaw cycles by preparing small aliquots

• Most antibodies are supplied in buffers containing preservatives such as sodium azide (typically 0.01-0.05%)

• Many formulations include stabilizers like glycerol (40%), BSA (0.05%), or similar proteins

• Some preparations are lyophilized and require reconstitution before use

It's critical to note that sodium azide, present in many antibody formulations, is considered hazardous and should be handled by trained staff only .

What are the basic properties of commercially available ASS1 recombinant monoclonal antibodies?

Key characteristics of ASS1 recombinant monoclonal antibodies include:

These properties impact the application suitability and performance characteristics of each antibody preparation .

What are the advantages of recombinant monoclonal antibodies over traditional monoclonal antibodies for ASS1 detection?

Recombinant monoclonal antibodies offer significant advantages for ASS1 research:

• Enhanced reproducibility: Manufactured using proprietary recombinant expression systems, ensuring consistent lot-to-lot performance

• Increased sensitivity: Higher affinity binding allows detection of lower abundance targets

• Superior specificity: Reduced cross-reactivity with other proteins

• Sustainable supply: Eliminated dependency on hybridomas or animals for production

• Animal-free production: Ethical advantage and reduced batch variability

• Defined sequence: Known antibody sequence enables genetic manipulation for specialized applications

• Improved stability: Generally more robust under various experimental conditions

• Reduced background: Cleaner signals in imaging and blotting applications

For example, the ZooMAb® system specifically produces antibodies with enhanced lot-to-lot consistency through proprietary recombinant expression, homogeneous purification, and precise dispensing .

How can I validate the specificity of an ASS1 recombinant monoclonal antibody for my research?

Comprehensive validation of ASS1 antibody specificity requires multiple approaches:

• Western blot analysis using multiple cell lines:

  • HeLa, HepG2, HEPG2, Hek293, THP-1, T47D cell lysates have been validated for ASS1 detection

  • Verify detection of a specific band at 47 kDa

• Tissue panel validation:

  • Human: liver, kidney, breast tissues

  • Animal models: rat liver/kidney, mouse liver/kidney tissues

• Negative controls:

  • ASS1 knockout cell lines when available

  • Secondary antibody-only controls

• Immunohistochemical correlation:

  • Compare staining patterns with published literature

  • Verify expected subcellular localization (typically cytoplasmic)

• Cross-platform validation:

  • Confirm results across multiple techniques (WB, IHC, IF)

• Peptide competition assay:

  • Pre-incubation with immunizing peptide should abolish specific signal

Validation images from manufacturers can provide guidance on expected results across different applications and sample types .

What protocols and parameters are optimal for using ASS1 antibodies in immunohistochemistry of formalin-fixed paraffin-embedded tissues?

For optimal IHC results with ASS1 antibodies in FFPE tissues:

Antigen Retrieval:

• Heat-mediated retrieval in EDTA buffer (pH 8.0) is consistently reported as effective

• Complete antigen unmasking is critical for accurate detection

Blocking and Antibody Incubation:

• Block with 10% goat serum to minimize background

• Primary antibody concentration: 1-2 μg/ml for most clones

• Incubation: Overnight at 4°C for optimal sensitivity

Detection Systems:

• Biotinylated secondary antibody followed by Streptavidin-Biotin-Complex (SABC)

• DAB as the chromogen provides good contrast

• Alternatively, fluorescent detection systems can be used for co-localization studies

Validated Tissue Types:

• Human tissues: liver, breast, prostate, colon (both normal and cancerous)

• Animal tissues: rat liver has shown good reactivity

Controls:

• Include positive control tissues with known ASS1 expression

• Include negative controls (primary antibody omission)

The detailed protocol validated by Boster Bio demonstrates successful IHC staining in various human cancer tissues using their monoclonal antibody at 2μg/ml concentration .

How can ASS1 antibodies be used to investigate the relationship between ASS1 expression and cancer progression?

ASS1 antibodies are valuable tools for cancer research due to ASS1's role as a potential tumor suppressor:

Methodological Approaches:

• Expression analysis across cancer types:

  • IHC screening of tissue microarrays reveals that ASS1 silencing or downregulation is common in various cancers including melanoma, prostate cancer, breast cancer, bladder cancer, mesothelioma, pancreatic cancer, and osteosarcomas

• Correlation with clinical outcomes:

  • Low ASS1 expression correlates with reduced metastasis-free survival in certain cancers

  • Kaplan-Meier analysis and Cox proportional hazard models can be used to assess relationships between ASS1 expression and patient prognosis

• Metabolic vulnerability assessment:

  • ASS1 deficiency creates a dependency on extracellular arginine for survival and proliferation

  • This metabolic vulnerability can be exploited therapeutically, as demonstrated with pegylated arginine deiminase (ADI-PEG20)

• Functional studies:

  • In breast cancer, ASS1 functions as a tumor suppressor

  • ASS1 activation by compounds like spinosyn A (SPA) suppresses breast tumor cell proliferation

• Mechanistic investigations:

  • ASS1 downregulation in cancer is associated with hypoxic conditions

  • Correlation studies with hypoxia markers (like CA9) can provide insights into regulatory mechanisms

Researchers have found that ASS1's enzymatic activity plays a crucial role in tumor suppression, particularly by affecting pyrimidine synthesis pathways .

What controls and experimental design considerations are essential when using ASS1 antibodies in cancer research?

Robust experimental design for ASS1 studies in cancer requires:

Control Samples:

• Paired normal/tumor tissues from the same patient

• Cancer cell lines with known ASS1 expression levels (high vs. low)

• ASS1 knockout cell lines as negative controls

• Isotype-matched control antibodies

Experimental Validation:

• Multiple antibody clones targeting different epitopes

• Cross-validation across different detection methods (WB, IHC, IF)

• Functional validation through gene silencing or overexpression

• Correlation with mRNA expression data

Data Analysis:

• Quantitative assessment of staining intensity

• Evaluation of subcellular localization

• Correlation with clinical parameters

• Statistical analysis accounting for confounding variables

Technical Considerations:

• Consistent sample processing and fixation

• Inclusion of technical replicates

• Blinded evaluation to prevent bias

• Standardized scoring systems for semi-quantitative analyses

Biological Context:

• Assessment of metabolic state (hypoxia, nutrient availability)

• Evaluation of related pathway components

• Consideration of tumor heterogeneity

For advanced studies, combining ASS1 antibody detection with metabolomic analyses can provide deeper insights into the functional consequences of ASS1 alterations in cancer .

What are the critical factors for successful Western blot detection of ASS1 protein?

Optimizing Western blot for ASS1 detection requires attention to several key parameters:

Sample Preparation:

• Validated sample types: HeLa, HepG2, Hek293, THP-1, and T47D cell lysates

• Animal tissues: rat/mouse liver and kidney tissues

• Recommended protein loading: 50 μg of whole cell/tissue lysate

• Use reducing conditions for optimal detection

Electrophoresis Conditions:

• 5-20% SDS-PAGE gels provide good resolution

• Running conditions: 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours

Transfer and Blocking:

• Transfer to nitrocellulose membrane at 150mA for 50-90 minutes

• Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

Antibody Incubation:

• Primary antibody concentration: 0.5 μg/mL to 1:1000 dilution (varies by manufacturer)

• Incubation: Overnight at 4°C for optimal sensitivity

• Secondary antibody: Anti-mouse/rabbit IgG-HRP at 1:10000 dilution

• Secondary incubation: 1.5 hours at room temperature

Detection:

• Enhanced chemiluminescent (ECL) detection systems work well

• Expected band size: 47 kD

Troubleshooting:

• If multiple bands appear, optimize antibody concentration and blocking conditions

• For weak signals, increase protein loading or primary antibody concentration

• High background may require more stringent washing or different blocking reagents

These parameters have been validated across multiple antibody preparations and should provide a starting point for optimization .

How can ASS1 antibodies be effectively used in flow cytometric applications?

For successful flow cytometry applications with ASS1 antibodies:

Sample Preparation:

• Cell fixation: Methods vary by antibody clone, but methanol fixation (100%, 5 minutes) works well for many antibodies

• Permeabilization: 0.1% Triton X-100 for 5 minutes to access intracellular ASS1

• Blocking: 1% BSA/10% normal serum/0.3M glycine in 0.1% PBS-Tween for 1 hour

Antibody Parameters:

• Recommended dilution ranges from 1:50 to 1:200 depending on the clone

• Successful detection demonstrated in multiple cell lines including Jurkat cells

• For conjugated antibodies (e.g., PE-conjugated), use appropriate compensation controls

Controls:

• Isotype control antibodies are essential

• Unstained cells for autofluorescence assessment

• Single-color controls for compensation

• When available, ASS1 knockout cells serve as excellent negative controls

Analysis Considerations:

• ASS1 is primarily an intracellular protein, requiring effective permeabilization

• Gating strategy should account for cell cycle phases, as metabolic enzyme expression may vary

• Consider co-staining with cell type markers for heterogeneous samples

Flow cytometric analysis allows for quantitative assessment of ASS1 expression at the single-cell level, enabling correlation with other cellular parameters .

What are the recommended protocols for using ASS1 antibodies in immunofluorescence and immunocytochemistry?

For optimal immunofluorescence and immunocytochemistry results:

Cell Preparation:

• Validated cell lines: MCF-7, NIH3T3, HeLa

• Fixation: 100% methanol (5 min) or formaldehyde/paraformaldehyde (4%)

• Permeabilization: 0.1% Triton X-100 (5 min)

Antigen Retrieval for ICC:

• Enzyme antigen retrieval reagent (e.g., IHC enzyme antigen retrieval reagent) for 15 mins

Blocking and Antibody Incubation:

• Block with 10% goat serum or 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween

• Primary antibody dilution: 1:50-1:1600 (optimize for each antibody)

• Primary antibody incubation: Often overnight at 4°C

• Secondary antibody: Fluorescently labeled (e.g., DyLight®488 Conjugated Goat Anti-Mouse IgG)

• Secondary antibody dilution: 1:100-1:1000

• Secondary incubation: 30 minutes at 37°C

Counterstaining and Mounting:

• Nuclear counterstain: DAPI works well with ASS1 cytoplasmic staining

• Mount with appropriate anti-fade mounting medium

• Seal edges with nail polish for long-term storage

Controls and Validation:

• Include negative controls (secondary antibody only)

• If available, ASS1 knockout cells provide excellent negative controls

• Validate staining pattern with published literature (typically cytoplasmic staining pattern)

The successful detection of ASS1 in MCF-7 cells using a mouse anti-ASS1 Antibody at 5μg/mL has been documented , providing a benchmark for optimization.

How do I select the most appropriate ASS1 recombinant monoclonal antibody clone for my specific research application?

Selection of the optimal ASS1 antibody clone requires consideration of multiple factors:

Application-Specific Performance:

• For Western blot: Clones like D4O4B, EPR12398, and 7I9 show strong performance

• For IHC-P: Clones R02-7A8, 2B10, and EPR12398 are well-validated

• For flow cytometry: 2B10 and EPR12398 have demonstrated efficacy

Species Compatibility:

• For human samples: All listed clones show reactivity

• For mouse/rat studies: Verify species cross-reactivity (7I9, EPR12398, 11F1-RA show multi-species reactivity)

Epitope Considerations:

• Antibodies targeting different regions may show variable results in certain applications

• C-terminal targeting antibodies (like D4O4B targeting residues surrounding Glu401)

• Middle region antibodies may have broader cross-reactivity

Format Options:

• Unconjugated for flexibility in detection methods

• Pre-conjugated versions (e.g., PE-conjugated) for direct detection

• Consider recombinant technology platforms (ZooMAb®, Picoband®, Hi-Affi™) for enhanced consistency

Validation Extent:

• Consider the breadth of validation data available

• Number of publications citing the antibody clone

• Range of validated applications and samples

Intended Research Focus:

• For cancer research: Clones validated in cancer tissues

• For metabolic studies: Antibodies validated in liver tissues

• For colocalization studies: Consider fluorophore-conjugated options

Cross-referencing manufacturer validation data with published literature using specific clones will help identify the most appropriate antibody for your research needs .

What methodologies can be used to study ASS1 as a potential therapeutic target using recombinant monoclonal antibodies?

ASS1 antibodies facilitate multiple approaches for therapeutic target validation:

Expression Profiling in Disease Models:

• IHC analysis to identify patient populations with altered ASS1 expression

• Correlation with clinical outcomes to establish prognostic value

• Identification of cancer subtypes with ASS1 deficiency that may respond to arginine deprivation therapies

Mechanistic Studies:

• Investigation of spinosyn A (SPA) and derivative LM-2I as ASS1 activators

• Analysis of the critical C13-C14 double bond in SPA and LM-2I and the Cys97 site in ASS1 for protein-small molecule interactions

• Assessment of ASS1 enzymatic activity enhancement following treatment

Functional Validation:

• Combining antibody-based detection with functional assays to correlate expression with activity

• Studies of arginine metabolism in ASS1-deficient versus ASS1-expressing cells

• Investigation of pyrimidine synthesis pathways affected by ASS1 modulation

Therapeutic Response Prediction:

• Antibody-based screening to identify candidates for arginine deprivation therapy

• Monitoring changes in ASS1 expression during treatment as a resistance mechanism

• Development of companion diagnostics for ASS1-targeted therapies

Combination Approaches:

• ASS1 detection combined with markers of tumor metabolism

• Integration with hypoxia markers like CA9 and MCT4

• Multi-parameter analysis of metabolic vulnerabilities