ASS1 Antibody

What is ASS1 and why is it important in biological research?

ASS1 (Argininosuccinate Synthetase 1) is an enzyme involved in the urea cycle, a sequence of biochemical reactions localized primarily in liver cells that processes excess nitrogen generated during protein metabolism . This nitrogen is used to create urea, which is subsequently excreted in urine . ASS1 plays a critical role in arginine biosynthesis, and its dysregulation has been implicated in various pathological conditions, particularly in cancer where ASS1 deficiency has been associated with poor prognosis . Recent research indicates that ASS1 also has important metabolic contributions to p53 regulation, and its loss can promote DNA damage and cell cycle progression, potentially contributing to cancer mutagenesis and adaptability .

What are the primary applications for ASS1 antibodies in laboratory research?

ASS1 antibodies are versatile reagents used across multiple experimental techniques. Based on validation data, the primary applications include:

These applications enable researchers to study ASS1 expression, localization, interactions, and function across different experimental systems .

Which cell lines and tissues have been validated for ASS1 antibody reactivity?

ASS1 antibodies have been validated across various biological samples:

The antibodies show cross-reactivity with human, mouse, and rat samples, making them suitable for comparative studies across species . When designing experiments, it's important to consider that reactivity may vary between antibody clones and that sample preparation methods might affect antibody performance in specific applications.

How should I optimize ASS1 antibody dilutions for different experimental applications?

Optimizing antibody dilution is crucial for obtaining specific signals while minimizing background noise. For ASS1 antibodies, follow these methodological approaches:

• Western Blot: Start with a 1:10000 dilution and adjust based on signal intensity. The recommended range is 1:5000-1:50000, but sample-dependent optimization is essential .

• Immunohistochemistry: Begin with a 1:200 dilution for paraffin sections. For ASS1 antibodies, a range of 1:50-1:500 is recommended . For optimal antigen retrieval, use TE buffer at pH 9.0 or alternatively citrate buffer at pH 6.0 .

• Immunofluorescence: Start with 1:400 dilution and adjust as needed within the 1:200-1:800 range .

• Flow Cytometry: Begin with manufacturer-recommended dilutions and perform a titration experiment with 3-4 dilutions to determine optimal concentration.

In all cases, include appropriate positive and negative controls to validate specificity. Each new lot of antibody should be re-optimized as there can be lot-to-lot variations in reactivity and optimal working concentrations.

What are the recommended storage conditions for maintaining ASS1 antibody stability?

To maintain ASS1 antibody activity and specificity over time, observe these storage protocols:

• Long-term storage: Store at -20°C. Most ASS1 antibodies remain stable for one year after shipment when properly stored .

• Short-term storage: For periods up to 1 month, storage at 4°C is acceptable .

• Avoid freeze-thaw cycles: Repeated freezing and thawing significantly degrades antibody quality. Aliquot antibodies before freezing if multiple uses are anticipated .

• Storage buffer: ASS1 antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Some preparations may contain 0.1% BSA in smaller volume sizes .

• Form and concentration: Most commercial ASS1 antibodies are supplied as liquid formulations at 1mg/ml concentration .

Proper storage and handling ensure consistent experimental results and extend the useful life of these valuable reagents.

How can I verify the specificity of my ASS1 antibody in experimental setups?

Verifying antibody specificity is critical for reliable research outcomes. Implement these methodological approaches:

• Knockdown/Knockout validation: Use siRNA/shRNA knockdown or CRISPR/Cas9 knockout of ASS1 in your experimental system, then confirm reduced or absent signal. Several publications have validated ASS1 antibodies using knockdown/knockout approaches .

• Multiple antibody validation: Use antibodies recognizing different epitopes of ASS1 (e.g., antibodies targeting AA 192-221 vs. AA 281-310) and compare staining patterns .

• Peptide competition: Pre-incubate the antibody with the immunizing peptide before application to your sample – specific signals should be blocked.

• Cross-reference with transcript levels: Compare protein expression detected by the antibody with mRNA levels from qPCR or RNA-seq data.

• Expected molecular weight verification: Confirm that the observed band in Western blot corresponds to the expected molecular weight of ASS1 (calculated: 47 kDa, commonly observed: 45 kDa) .

• Positive and negative control tissues: Include samples known to express (liver, kidney) or lack ASS1 in your experiments.

By implementing these validation strategies, you can ensure the reliability of your experimental results with ASS1 antibodies.

How can ASS1 antibodies be used to study arginine metabolism in cancer research?

ASS1 antibodies serve as powerful tools for investigating arginine metabolism in cancer, particularly in the context of arginine auxotrophy and targeted therapies:

• Stratification of tumors for arginine deprivation therapy: IHC with ASS1 antibodies can identify ASS1-deficient tumors that may be susceptible to arginine deprivation therapies like ADI-PEG20 . Research shows that ASS1-deficient cell lines respond to arginine starvation with decreased proliferation, while ASS1-expressing cells are resistant .

• Monitoring resistance mechanisms: Western blot analysis using ASS1 antibodies can track the re-expression of ASS1 in initially deficient tumors during treatment. As demonstrated in sarcoma and melanoma cell lines, long-term ADI-PEG20 treatment leads to increased ASS1 expression as a primary resistance mechanism .

• Investigating metabolic adaptation: Through co-immunoprecipitation experiments with ASS1 antibodies, researchers can identify protein-protein interactions that change during metabolic adaptation to arginine deprivation.

• Subcellular localization studies: Immunofluorescence with ASS1 antibodies can reveal changes in enzyme localization during stress responses, providing insights into non-canonical functions of ASS1.

• Cancer mutagenesis studies: Recent research indicates that loss of ASS1 contributes to DNA damage and promotes cell cycle progression, potentially contributing to cancer mutagenesis and adaptability . ASS1 antibodies enable researchers to correlate ASS1 expression with markers of DNA damage and cell cycle regulation.

These applications highlight how ASS1 antibodies contribute to understanding cancer metabolism and developing targeted therapeutic approaches.

What experimental controls should be included when investigating ASS1 expression in relation to p53 regulation?

• Cell line selection controls:

  • Include both p53 wild-type and p53-null or mutant cell lines to distinguish p53-dependent and independent effects

  • Use cell lines with different baseline ASS1 expression (positive and negative)

• Expression modulation controls:

  • For ASS1 knockdown/knockout experiments, include scrambled siRNA or empty vector controls

  • When overexpressing ASS1, use enzymatically inactive mutants to distinguish catalytic from structural functions

• Subcellular fractionation quality controls:

  • Verify fractionation purity using markers for nuclear (e.g., Lamin B1) and cytosolic (e.g., GAPDH) compartments

  • Confirm that observed changes in ASS1 localization aren't artifacts of isolation procedures

• p53 activation controls:

  • Include both DNA damage-inducing agents (e.g., doxorubicin) and non-genotoxic p53 activators

  • Monitor canonical p53 targets (p21, MDM2) alongside ASS1 to confirm p53 pathway activation

• Metabolic state controls:

  • Since ASS1 functions in arginine metabolism, control for arginine availability in experimental media

  • Monitor related metabolic enzymes in the urea cycle to distinguish ASS1-specific effects

Recent research suggests that ASS1 metabolically contributes to nuclear and cytosolic p53 regulation . Therefore, proper controls for both metabolic functions and protein interactions are necessary to delineate the precise mechanisms involved.

How can I troubleshoot inconsistent ASS1 antibody staining patterns in tissue microarrays?

Inconsistent staining in tissue microarrays (TMAs) can compromise research results. Address these issues with systematic troubleshooting:

• Fixation and processing variables:

  • Standardize fixation time across samples; overfixation can mask epitopes

  • Different tissue sources may have varied processing protocols; document and account for these differences

  • For ASS1 specifically, test both TE buffer (pH 9.0) and citrate buffer (pH 6.0) for antigen retrieval, as both have been validated

• Antibody-specific factors:

  • Determine optimal concentration through titration experiments (1:50-1:500 range for IHC)

  • Consider epitope location – antibodies targeting different regions (e.g., AA 192-221 vs. AA 281-310) may show different sensitivities to fixation

  • Batch effects can occur; use the same lot number across a study when possible

• Biological variables:

  • ASS1 expression is highly tissue-dependent; liver typically shows strong expression while some cancers are ASS1-deficient

  • Heterogeneous expression within tumors is common; multiple cores per tumor may be needed

  • Cell-type specific expression can cause apparent inconsistency in mixed cell populations

• Technical approaches:

  • Implement automated staining platforms to improve consistency

  • Use digital pathology quantification to objectively assess staining intensity

  • Consider multiplex IHC to simultaneously detect ASS1 alongside cell-type markers

• Validation approaches:

  • Correlate IHC findings with other methods (Western blot, qPCR) from the same samples

  • Include known positive controls (e.g., normal liver) on each TMA slide

By methodically addressing these factors, researchers can achieve more consistent and interpretable ASS1 staining patterns in TMAs.

How does ASS1 expression change in response to arginine deprivation, and how can antibodies help monitor this process?

ASS1 expression demonstrates dynamic regulation in response to arginine availability, which can be monitored using antibodies:

• Short-term vs. long-term adaptation: ASS1-deficient cells initially show modest increases in ASS1 expression upon arginine deprivation, but long-term exposure leads to substantial upregulation. Western blot using ASS1 antibodies can track this progression, as demonstrated in studies with ADI-PEG20 treatment .

• Transcriptional regulation: c-Myc-dependent re-expression of ASS1 is a primary mechanism of resistance to arginine starvation . Combining ASS1 antibodies with c-Myc antibodies in co-immunostaining experiments can reveal temporal relationships between these factors.

• Cell-type specific responses: Different cell types respond variably to arginine deprivation. For instance, human sarcoma cell lines (SKLMS1, SKUT1) and melanoma cells (SKMEL2) show distinct patterns of ASS1 re-expression during adaptation to arginine starvation . Immunofluorescence with ASS1 antibodies can characterize these cell-specific differences.

• Phenotypic consequences: Arginine starvation in ASS1-negative cells leads primarily to cytostatic rather than cytotoxic responses . Time-course experiments combining ASS1 antibody staining with proliferation and cell death markers can elucidate these relationships.

• Therapeutic implications: Monitoring ASS1 expression using antibodies during arginine deprivation therapy provides crucial information about developing resistance. In experimental models, Long-Term ADI-PEG20 Treatment (LTAT) resistant cells show growth rates similar to untreated wild-type cells despite continued arginine deprivation .

This dynamic regulation underscores the importance of using ASS1 antibodies to monitor expression changes in both research and clinical contexts.

What is the relationship between ASS1 deficiency and DNA damage in cancer cells?

Recent research has uncovered important connections between ASS1 deficiency and genomic instability:

• Mechanistic relationship: Prolonged loss of ASS1 promotes DNA damage in colon cancer cells and fibroblasts . This connection likely involves metabolic perturbations that affect nucleotide balance and DNA repair processes.

• Cell cycle effects: ASS1 deficiency promotes cell cycle progression despite DNA damage, potentially contributing to accumulation of mutations and cancer adaptability . Immunofluorescence co-staining using ASS1 antibodies alongside cell cycle markers (e.g., Ki-67) and DNA damage markers (e.g., γH2AX) can visualize these relationships.

• p53 connection: ASS1 metabolically contributes to both nuclear and cytosolic p53 regulation . This interaction suggests a feedback mechanism where ASS1 deficiency affects p53 function, which normally would halt cell cycle in response to DNA damage.

• Metabolic basis: In ASS1-deficient cells, cytosolic aspartate is diverted toward pyrimidine synthesis, supporting proliferation but potentially causing nucleotide imbalance that contributes to mutagenesis . Combining ASS1 antibody detection with metabolic profiling can help establish these connections.

• Clinical relevance: The link between ASS1 deficiency, DNA damage, and mutagenesis may partly explain why ASS1-negative tumors often have poor prognosis . ASS1 antibodies are therefore valuable prognostic tools in cancer pathology.

These findings highlight the importance of ASS1 not only as a metabolic enzyme but also as a factor in genome stability and cancer progression.

What are the key differences between monoclonal and polyclonal ASS1 antibodies for research applications?

Understanding the differences between monoclonal and polyclonal ASS1 antibodies helps researchers select the optimal reagent for specific applications:

When selecting between these options, consider:

• For detecting low levels of ASS1, polyclonal antibodies may provide greater sensitivity

• For applications requiring high specificity and reproducibility across experiments, monoclonal antibodies may be preferable

• For confirming results, using both types that recognize different epitopes provides stronger validation

How can I quantitatively compare ASS1 expression levels across different experimental conditions?

Accurate quantitative comparison of ASS1 expression requires methodological rigor and appropriate analytical approaches:

• Western Blot Quantification:

  • Use gradient loading to ensure measurements fall within the linear detection range

  • Include recombinant ASS1 protein standards at known concentrations

  • Normalize to multiple housekeeping proteins (e.g., β-actin, GAPDH, tubulin)

  • Use digital imaging and analysis software with background subtraction

  • For ASS1, look for the expected 45 kDa band

• Flow Cytometry Approaches:

  • Use median fluorescence intensity (MFI) ratios relative to isotype controls

  • Include calibration beads with known antibody binding capacity

  • Perform parallel analysis of known ASS1-positive and negative cell lines

  • Consider dual staining with antibodies targeting different ASS1 epitopes

• Immunohistochemistry Quantification:

  • Use digital pathology platforms for objective scoring

  • Implement H-score (intensity × percentage positive cells) methodology

  • Include reference tissues with established ASS1 expression levels on each slide

  • Consider automated multispectral imaging systems for more precise quantification

• Transcript-Protein Correlation:

  • Correlate protein levels determined by ASS1 antibodies with mRNA levels

  • Assess protein stability through cycloheximide chase experiments

  • Compare relative changes at protein and mRNA levels to identify post-transcriptional regulation

• Validation Across Methods:

  • Compare results across multiple quantitative techniques

  • Use absolute quantification methods (e.g., selected reaction monitoring mass spectrometry) for calibration

  • Consider the dynamic range limitations of each method

By implementing these approaches, researchers can achieve reliable quantitative comparisons of ASS1 expression across experimental conditions.

What are the critical considerations when selecting an ASS1 antibody for studying protein-protein interactions?

Selecting appropriate ASS1 antibodies for protein interaction studies requires careful consideration of several factors:

• Epitope location awareness:

  • Choose antibodies whose epitopes don't interfere with interaction domains

  • For co-immunoprecipitation, avoid antibodies targeting regions involved in protein-protein binding

  • Different antibodies (e.g., those targeting AA 192-221 vs. AA 281-310) may yield different interaction profiles

• Antibody format requirements:

  • For co-immunoprecipitation, select antibodies that have been validated for IP applications

  • For proximity ligation assays, ensure both primary antibodies are from different host species

  • For FRET studies, consider directly conjugated antibodies with appropriate fluorophores

• Binding characteristics:

  • Higher affinity antibodies generally perform better in detecting weak or transient interactions

  • Non-denaturing conditions are usually required; confirm antibody works in native conditions

  • Validate specificity under the exact buffer conditions used for interaction studies

• Validation approaches:

  • Verify that immunoprecipitation with the selected antibody enriches ASS1 (Western blot)

  • Confirm the antibody doesn't cross-react with known or suspected interaction partners

  • Use multiple antibodies targeting different epitopes to validate interactions

• Technical considerations:

  • For ASS1 co-immunoprecipitation, typical protocols recommend 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

  • Include appropriate negative controls (isotype-matched antibodies, ASS1-negative cells)

  • Consider crosslinking antibodies to beads to prevent antibody contamination in mass spectrometry analysis

By carefully selecting and validating ASS1 antibodies for protein interaction studies, researchers can minimize artifacts and generate reliable data about ASS1's interaction network.

What emerging applications of ASS1 antibodies are being developed in cancer metabolism research?

The field of ASS1 research is evolving rapidly, with several emerging applications of ASS1 antibodies in cancer metabolism:

• Liquid biopsy development: Researchers are exploring the use of ASS1 antibodies to detect circulating tumor cells with specific metabolic phenotypes, potentially allowing non-invasive monitoring of ASS1 status during arginine deprivation therapy.

• Therapeutic response prediction: Combining ASS1 antibody-based tissue analysis with metabolomic profiling to develop predictive algorithms for response to arginine deprivation therapies, enabling precision medicine approaches.

• Multiparameter imaging: Advanced multiplexed immunofluorescence techniques using ASS1 antibodies alongside markers of metabolic state, DNA damage, and cell cycle to create comprehensive spatial maps of tumor metabolism.

• Metabolic crosstalk visualization: Using ASS1 antibodies in combination with antibodies against other metabolic enzymes to visualize enzyme proximity and potential metabolon formation in different cellular compartments.

• ASS1-p53 interaction studies: Given recent findings linking ASS1 to p53 regulation , antibodies are being used to investigate the subcellular compartmentalization of this relationship and its impact on cancer cell adaptation.

These emerging applications highlight the continuing importance of ASS1 antibodies in uncovering the complex roles of this enzyme in cancer biology and metabolism.

How can researchers validate and compare results obtained with different commercial ASS1 antibodies?

Systematic validation and comparison of different commercial ASS1 antibodies is essential for research reproducibility:

• Panel testing approach:

  • Test multiple antibodies (e.g., Proteintech 16210-1-AP, Prospec ANT-639, antibodies-online ABIN390810) in parallel on the same samples

  • Include positive controls (liver tissue, ASS1-expressing cell lines) and negative controls (ASS1-knockout cells)

  • Document lot numbers, dilutions, and detailed protocols for future reference

• Cross-application validation:

  • Compare antibody performance across multiple applications (WB, IHC, IF)

  • Determine if antibodies show consistent relative expression patterns even if absolute signals differ

  • Identify application-specific strengths of each antibody

• Epitope mapping considerations:

  • Compare antibodies targeting different regions (N-terminal, central, C-terminal)

  • Central region antibodies (AA 192-221) may detect different conformational states than C-terminal antibodies (AA 281-310)

  • Document any discrepancies that might reveal biologically relevant information about protein processing or interactions

• Quantitative comparisons:

  • Use recombinant ASS1 protein standards to compare absolute sensitivity

  • Determine linear detection ranges for each antibody

  • Assess signal-to-noise ratios across a concentration gradient

• Biological validation:

  • Correlate antibody staining with genetic manipulation of ASS1 (overexpression, knockdown)

  • Compare antibody detection with orthogonal measures (mass spectrometry, enzymatic activity)

  • Evaluate concordance between protein detection and mRNA levels