ASN1 Antibody

What is ASNS and what is its biological significance?

Asparagine synthetase (ASNS) is an enzyme that catalyzes the synthesis of asparagine and glutamate using glutamine and aspartate as substrates. The ASNS gene is highly regulated by the nutritional status of the cell, making it an important marker for cellular metabolic states . ASNS plays critical roles in various biological processes including amino acid metabolism, cellular adaptation to nutrient availability, and cancer cell survival mechanisms. The protein has several isoforms with molecular weights ranging between 55-64 kDa, which should be considered when analyzing experimental results .

What are the primary applications for ASNS antibodies in research?

ASNS antibodies are versatile tools applicable across multiple experimental techniques. According to validation data, ASNS antibodies can be reliably used in Western blotting (WB), immunohistochemistry (IHC), immunofluorescence/immunocytochemistry (IF/ICC), immunoprecipitation (IP), and ELISA applications . The antibody has been validated in numerous published studies with over 50 publications utilizing it for Western blotting and 10 publications for immunohistochemistry applications . This demonstrates the antibody's reliability across different experimental contexts and research questions.

What is the recommended starting point for working with ASNS antibodies?

Researchers new to working with ASNS antibodies should begin with a dilution range test. For immunohistochemistry, start with dilutions between 1:50-1:500 . For immunofluorescence or immunocytochemistry, similar dilution ranges (1:50-1:500) are appropriate . For immunoprecipitation, use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate . Always include appropriate positive controls such as HeLa cells for IP, human retinoblastoma or gliomas tissue for IHC, and K-562 cells for IF/ICC based on validated reactivity data .

What storage conditions should be maintained for ASNS antibodies?

ASNS antibodies should be stored at -20°C for long-term stability . The antibody solution contains PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For antibodies provided in small volumes (20μl), they may contain 0.1% BSA as a stabilizer . The antibody remains stable for one year after shipment when stored properly. Aliquoting is generally unnecessary for -20°C storage, which simplifies laboratory management of the reagent . Avoiding repeated freeze-thaw cycles is recommended to maintain antibody functionality.

How should samples be prepared for optimal ASNS detection?

Sample preparation varies by application. For Western blotting, standard denaturing conditions with SDS-sample buffer are effective, as verified by customer reviews showing specific bands for ASNS in human RCC4 kidney cancer cell lysates . For immunofluorescence, standard fixation protocols have been successful in visualizing ASNS in mouse kidney samples, with good results using DAPI counterstaining for nuclear visualization . For immunohistochemistry, the choice of antigen retrieval buffer significantly impacts results, with TE buffer (pH 9.0) generally providing optimal staining for human tissues .

How does ASNS function in cancer metabolism and what research applications are emerging?

ASNS has emerged as a critical player in cancer metabolism research. Recent studies have demonstrated that ASNS couples mitochondrial respiration to ATF4 activity and tumor growth . Additionally, ASNS has been implicated in cellular adaptation to glutamine depletion, which is relevant to understanding metabolic reprogramming in cancer cells . Research applications include studying how glutamine depletion regulates Slug to promote EMT and metastasis in pancreatic cancer . ASNS antibodies are valuable tools for investigating these mechanisms through protein expression analysis, co-localization studies, and protein-protein interaction experiments.

What is the relationship between ASNS and p53 signaling?

Recent research has revealed that p53 plays a crucial role in controlling aspartate-asparagine homeostasis, which directly influences LKB1 activity and modulates cell survival . This relationship suggests that ASNS antibodies can be valuable tools in research investigating metabolic control mechanisms and their connection to tumor suppressor pathways. Researchers can design experiments using ASNS antibodies to explore how metabolic stress affects p53 activation and subsequent changes in asparagine synthesis, providing insights into potential therapeutic targets for cancer treatment.

How can ASNS antibodies be used in knockdown/knockout validation studies?

ASNS antibodies have been extensively used in knockdown and knockout studies, with at least 15 published papers utilizing this approach . These studies are critical for validating antibody specificity and for examining the functional consequences of ASNS depletion. When designing such experiments, researchers should include appropriate controls and consider the potential compensatory mechanisms that may arise in response to ASNS depletion. The antibody's proven track record in KD/KO studies makes it a reliable tool for validating genetic manipulation experiments targeting the ASNS gene.

What are common issues encountered with ASNS antibody staining and how can they be resolved?

When using ASNS antibodies in immunohistochemistry, non-specific background staining can occur. This may be resolved by:

• Optimizing antibody dilution (try the recommended range of 1:50-1:500)

• Testing different antigen retrieval methods (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

• Increasing blocking time or concentration

• Using sample-specific optimized protocols as noted in the antibody documentation

For Western blotting applications, multiple bands or unexpected molecular weights may be observed due to the presence of ASNS isoforms (55-64 kDa range) . Longer exposure times may be necessary to detect lower abundance isoforms.

How should researchers address batch-to-batch variability when working with ASNS antibodies?

Batch-to-batch variability can be addressed by:

• Always testing new antibody lots against a previous lot that worked well

• Maintaining consistent positive controls across experiments

• Using the same dilution series to determine optimal working concentration for each new lot

• Documenting lot numbers and performance characteristics for reproducibility

• Following the manufacturer's specific protocols for each application (WB, IHC, IF, IP)

What controls are essential when working with ASNS antibodies?

Essential controls for ASNS antibody experiments include:

• Positive tissue controls: Human retinoblastoma tissue and human gliomas tissue have been validated for IHC applications

• Positive cell line controls: HeLa cells for IP applications and K-562 cells for IF/ICC applications

• Negative controls: Secondary antibody-only controls to assess background

• Isotype controls: Using matched isotype (Rabbit IgG) at equivalent concentrations

• Knockdown/knockout controls: Especially valuable given the extensive validation in KD/KO studies

How should ASNS expression data be interpreted across different cancer types?

When interpreting ASNS expression data across cancer types, researchers should consider:

• Baseline expression varies significantly between tissue types

• ASNS has been specifically studied in retinoblastoma and gliomas with validated antibody reactivity

• Expression patterns should be interpreted in the context of metabolic status and glutamine availability

• Consider that multiple isoforms (55-64 kDa) may be present and differently expressed

• Correlation with patient outcomes requires careful statistical analysis with appropriate cohort sizes

Recent publications have demonstrated ASNS involvement in pancreatic cancer metastasis, making it an important biomarker for cancer progression studies .

What is the significance of subcellular localization in ASNS studies?

ASNS subcellular localization can provide important insights into its functional state and regulatory mechanisms. Immunofluorescence studies have successfully detected ASNS in various cell types including K-562 cells . When analyzing subcellular localization:

• Compare distribution patterns under normal versus stressed conditions

• Assess co-localization with other metabolic enzymes or organelle markers

• Consider changes in localization following drug treatments or metabolic perturbations

• Utilize Z-stack imaging for three-dimensional distribution analysis

• Quantify nuclear versus cytoplasmic distribution ratios for comparative studies

How do ASNS expression levels correlate with glutamine metabolism in experimental models?

Recent research indicates that ASNS plays a critical role in cellular adaptation to glutamine depletion . When analyzing the relationship between ASNS expression and glutamine metabolism:

Studies have shown that filamentous GLS1 promotes ROS-induced apoptosis upon glutamine deprivation via insufficient asparagine synthesis, highlighting the complex interplay between these metabolic pathways .

How is ASNS being investigated in the context of therapeutic resistance?

ASNS has emerged as an important factor in therapeutic resistance, particularly in the context of L-asparaginase treatment. Recent studies have identified that ZBTB1 regulates asparagine synthesis and leukemia cell response to L-asparaginase . Researchers investigating therapeutic resistance should:

• Examine ASNS expression before and after treatment exposure

• Correlate ASNS levels with treatment outcomes

• Consider combination approaches targeting ASNS regulatory pathways

• Investigate the relationship between ASNS expression and other resistance mechanisms

• Explore how metabolic adaptations involving ASNS contribute to therapeutic escape

What role does ASNS play in epithelial-mesenchymal transition (EMT)?

Emerging research has revealed that glutamine depletion regulates Slug to promote EMT and metastasis in pancreatic cancer, with ASNS playing a key role in this process . When investigating ASNS in EMT:

• Assess how ASNS expression correlates with EMT markers (E-cadherin, N-cadherin, Vimentin)

• Analyze ASNS regulation during different stages of the metastatic cascade

• Examine how metabolic stress influences ASNS-dependent EMT processes

• Consider the impact of ASNS inhibition on cell migration and invasion capabilities

• Investigate potential transcriptional regulators controlling both ASNS expression and EMT programs