AGXT2 Antibody

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Description

Definition and Purpose of AGXT2 Antibody

AGXT2 antibodies are monoclonal or polyclonal reagents that bind specifically to the AGXT2 protein, a pyridoxal phosphate-dependent mitochondrial aminotransferase. AGXT2 catalyzes the transamination of ADMA, a nitric oxide synthase inhibitor linked to hypertension and endothelial dysfunction . These antibodies are pivotal for:

  • Detecting AGXT2 expression in tissues (e.g., kidney, liver) via Western blot (WB) or immunohistochemistry (IHC) .

  • Studying AGXT2's enzymatic activity and its impact on methylarginine metabolism .

  • Validating AGXT2 knockout (KO) or overexpression models in preclinical studies .

Research Applications and Findings

AGXT2 antibodies have been instrumental in advancing understanding of AGXT2’s physiological and pathological roles:

Cardiovascular Research

  • Hypertension Models: AGXT2 knockout mice exhibit elevated ADMA, reduced NO bioavailability, and hypertension . Antibodies confirmed AGXT2 absence in renal mitochondria, correlating with increased plasma ADMA (25%) and systolic blood pressure (10–20 mmHg) .

  • Vascular Protection: Overexpression of AGXT2 in transgenic mice lowered ADMA by 15%, reduced aortic remodeling, and improved endothelial function .

Renal Disease

  • Acute Kidney Injury (AKI): AGXT2 expression is downregulated in AKI models, linked to ADMA accumulation and renal dysfunction . Immunohistochemistry using AGXT2 antibodies revealed reduced protein levels in injured kidneys .

Metabolic Studies

  • Enzyme Activity: AGXT2 antibodies validated mitochondrial localization and transamination activity in kidney isolates, showing ADMA metabolism even at low intracellular concentrations (1–100 μM) .

Table 2: Product Comparison

Product IDHostApplicationsKey Validations
66602-1-IgMouseWB, IHC, CoIPConfirmed knockout validation
ab231815RabbitWB, IHC-P, ELISADetects human AGXT2 isoforms
ab232803RabbitWB, IHC-PValidated in mouse liver/kidney

Key Considerations:

  • Species Specificity: Mouse-derived clones (e.g., 66602-1-Ig) are optimal for murine studies, while rabbit polyclonals (e.g., ab231815) suit human tissue .

  • Dilution Ranges: WB typically uses 1:5,000–1:50,000 dilutions; IHC requires 1:500–1:2,000 .

Validation and Quality Control

  • Western Blot: AGXT2 antibodies consistently detect a 57 kDa band in human/mouse kidney and liver lysates .

  • Immunohistochemistry: Strong mitochondrial staining in renal tubules and hepatocytes .

  • Knockout Validation: Absence of signal in AGXT2-deficient mice confirms antibody specificity .

Emerging Insights and Clinical Relevance

  • Genetic Polymorphisms: AGXT2 SNPs (e.g., rs37369) correlate with elevated ADMA and hypertension risk, highlighting its role in cardiovascular disease .

  • Therapeutic Potential: AGXT2 upregulation mitigates ADMA-induced vascular damage, suggesting drug-target potential .

Product Specs

Buffer
Preservative: 0.03% Proclin 300
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Form
Liquid
Lead Time
Typically, we are able to ship products within 1-3 business days after receiving your order. Delivery times may vary depending on the mode of purchase or location. For specific delivery timeframes, please consult your local distributor.
Synonyms
AGXT2 antibody; AGT2Alanine--glyoxylate aminotransferase 2 antibody; mitochondrial antibody; AGT 2 antibody; EC 2.6.1.44; antibody; R)-3-amino-2-methylpropionate--pyruvate transaminase antibody; EC 2.6.1.40 antibody; Beta-ALAAT II antibody; Beta-alanine-pyruvate aminotransferase antibody; D-AIBAT antibody
Target Names
AGXT2
Uniprot No.

Target Background

Function
AGXT2 is an enzyme capable of metabolizing asymmetric dimethylarginine (ADMA) through transamination to alpha-keto-delta-(NN-dimethylguanidino) valeric acid (DMGV). ADMA is a potent inhibitor of nitric-oxide (NO) synthase, and this activity provides a mechanism through which the kidney regulates blood pressure.
Gene References Into Functions
  1. Our research involved purifying human AGXT2 from tissues of AGXT2 transgenic mice. We demonstrated its ability to metabolize homoarginine to 6-guanidino-2-oxocaproic acid (GOCA). After incubating HepG2 cells overexpressing AGXT2 with isotope-labeled homoarginine-d4, we were able to detect labeled GOCA in the medium. PMID: 27752063
  2. A study conducted in a well-characterized rheumatoid arthritis population did not reveal an association between serum concentrations of dimethylarginines and genetic variants of the AGXT2 gene. PMID: 28357606
  3. The AGXT2 rs37369 polymorphism is associated with an increased risk for chronic heart failure, potentially due to distinct disparities in asymmetric dimethylarginine degradation. PMID: 27423328
  4. Single nucleotide polymorphism in the AGXT2 gene is linked to renal dysfunction in patients with chronic heart failure. PMID: 28942034
  5. The Alanine-glyoxylate aminotransferase 2 p.V498L polymorphism is associated with both paroxysmal and chronic forms of atrial fibrillation in coronary angiographic patients without structural heart disease in ultrasound, and earlier age at onset of ischemic stroke in patients undergoing exercise stress testing. PMID: 26984639
  6. The AGXT2 rs37369 polymorphism is associated with an increased risk for coronary heart disease (CHD) in smokers and individuals with diabetes mellitus. PMID: 24834905
  7. Our findings suggest that the AGXT2 genotype may be a significant factor contributing to atherosclerosis. PMID: 25620171
  8. This study concluded that the AGXT2 gene is not associated with schizophrenia in Japanese subjects. PMID: 24727203
  9. Single nucleotide polymorphisms (SNPs) in AGXT2 influence both plasma and urinary BAIB levels. PMID: 24586340
  10. AGXT2 plays a crucial role in SDMA metabolism in humans and may also have an unexpected role in the autonomic nervous system regulation of cardiac function. PMID: 24159190
  11. Alanine-glyoxylate aminotransferase-2 metabolizes endogenous methylarginines, regulates NO, and controls blood pressure. PMID: 23023372
  12. Mitochondrially localized human AGXT2 effectively metabolizes ADMA in vivo, resulting in decreased ADMA levels and improved endothelial NO production. PMID: 20018850
  13. This publication discusses the cloning of the rat gene. PMID: 7592550

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Database Links

HGNC: 14412

OMIM: 210100

KEGG: hsa:64902

STRING: 9606.ENSP00000231420

UniGene: Hs.34494

Protein Families
Class-III pyridoxal-phosphate-dependent aminotransferase family
Subcellular Location
Mitochondrion.

Q&A

What is AGXT2 and what are its primary functions?

AGXT2 is a mitochondrially-localized aminotransferase with broad substrate specificity. It plays several important metabolic roles:

  • Catalyzes the conversion of glyoxylate to glycine using alanine as an amino donor

  • Metabolizes D-beta-aminoisobutyric acid to generate 2-methyl-3-oxopropanoate and alanine

  • Transfers amino groups from beta-alanine to pyruvate, yielding L-alanine and 3-oxopropanoate

  • Most significantly, metabolizes asymmetric dimethylarginine (ADMA), which is a potent inhibitor of nitric oxide (NO) synthase

Through its ADMA-metabolizing activity, AGXT2 provides a mechanism by which the kidney can regulate blood pressure and endothelial function . AGXT2 functions in both glyoxylate and dicarboxylate metabolism pathways as well as broader amino acid metabolism pathways, maintaining the balance between glycolate and glycine levels essential for normal cellular function .

What is the cellular localization of AGXT2 and how does this impact antibody selection?

AGXT2 is primarily localized in mitochondria, containing a 41-amino acid N-terminal mitochondrial cleavage sequence . This mitochondrial localization has been confirmed through confocal microscopy studies after expression of FLAG-tagged AGXT2 .

When selecting antibodies for AGXT2 detection, researchers should consider:

  • For immunohistochemistry or immunofluorescence: Antibodies should be able to penetrate fixed and permeabilized cells to access mitochondrial proteins

  • For detecting the mature protein: Antibodies targeting regions downstream of the 41-amino acid N-terminal sequence should be preferred

  • For subcellular fractionation experiments: Positive mitochondrial markers should be used alongside AGXT2 antibodies to confirm proper fractionation

In experimental systems using tagged AGXT2 (such as FLAG-tagged constructs), researchers can alternatively use high-quality commercial anti-tag antibodies for detection, as demonstrated in several studies .

How should AGXT2 antibodies be validated before experimental use?

Thorough validation of AGXT2 antibodies is essential for obtaining reliable results. Based on research practices, a comprehensive validation approach should include:

  • Specificity testing:

    • Western blot analysis using positive control tissues (kidney and liver lysates)

    • Comparison of results in wild-type versus AGXT2-knockout or AGXT2-overexpressing systems

    • Peptide competition assays to confirm epitope specificity

  • Cross-reactivity assessment:

    • Testing antibody reactivity across species if cross-species experimentation is planned

    • Confirming lack of cross-reactivity with related aminotransferases such as AGXT1

  • Application-specific validation:

    • For Western blot: Confirm single band at expected molecular weight (~52 kDa for mature protein)

    • For IHC/IF: Verify localization pattern matches expected mitochondrial distribution

    • For IP: Confirm enrichment of target protein and co-immunoprecipitation of known binding partners

  • Positive controls:

    • Include kidney and liver tissues, where AGXT2 is known to be highly expressed

    • Consider engineered cells with FLAG-tagged AGXT2 as additional positive controls

What are the best tissue types for positive and negative controls when testing AGXT2 antibodies?

Based on experimental findings from multiple studies, the following tissues serve as appropriate controls:

Positive control tissues:

  • Kidney (primary expression site)

  • Liver (primary expression site)

  • Particularly renal tubules, which show strong AGXT2 expression by IHC staining

Negative/low expression controls:

  • Brain tissue (typically low expression of AGXT2)

  • Samples from verified AGXT2 knockout models

  • Tissues pretreated with validated AGXT2-blocking peptides

For immunohistochemistry specifically, renal tubules show stronger staining than glomeruli, which can serve as an internal reference for staining intensity gradation . In a study examining AGXT2 in acute kidney injury, immunohistochemistry revealed that "AGXT2 was mainly expressed in the renal tubules," making this tissue particularly valuable for antibody validation .

How should experimental design address potential artifacts when studying AGXT2 overexpression?

When designing experiments involving AGXT2 overexpression, several considerations must be addressed to avoid experimental artifacts:

  • Expression system selection:

    • Viral vector selection is crucial. Studies have successfully used adenoviral vectors for transient expression, but these may induce immune responses in longer-term studies

    • For stable expression, transgenic mouse models using CAG promoters have proven effective

  • Transgene verification protocols:

    • Employ multiple methods to confirm expression:

      • qPCR using transgene-specific primers (e.g., human AGXT2-specific primers for human transgenes in mouse models)

      • Western blot using antibodies specific to transgenic protein or epitope tags

      • Enzymatic activity assays measuring AGXT2-specific metabolic products

  • Controlling localization:

    • Verify mitochondrial localization of overexpressed protein to ensure proper function

    • For epitope-tagged constructs, confirm tag placement doesn't interfere with mitochondrial targeting

    • Researchers have successfully used C-terminal FLAG tags without disrupting localization

  • Functional validation:

    • Measure ADMA levels in plasma/tissue to confirm functional relevance of overexpression

    • Quantify AGXT2-specific metabolites like ADGV (asymmetric dimethylguanidino valeric acid)

    • In vascular studies, confirm expected phenotypic changes (improved endothelial function)

An effective approach demonstrated in research involves crossing AGXT2 transgenic mice with disease models (e.g., DDAH1 knockout mice) to establish the protective effects of AGXT2 overexpression against specific pathologies .

What methodological approaches can resolve contradictory AGXT2 expression data across different experimental systems?

When faced with contradictory AGXT2 expression data across different experimental systems, researchers should implement the following methodological approaches:

  • Standardized quantification methods:

    • Employ absolute quantification using standard curves with recombinant AGXT2 protein

    • Use digital PCR for more precise transcript quantification

    • Implement rigorously validated reference genes for relative quantification

  • Multi-level expression analysis:

    • Compare mRNA expression (qPCR) with protein levels (Western blot, ELISA)

    • Assess enzyme activity using metabolic product analysis (e.g., ADGV levels)

    • Perform in situ hybridization alongside IHC to correlate localization patterns

  • Consider post-transcriptional/post-translational regulation:

    • Examine miRNA regulation of AGXT2 expression

    • Investigate protein stability and turnover rates

    • Assess potential splice variants with isoform-specific primers

  • Standardize experimental conditions:

    • Control for circadian variations in expression

    • Account for dietary factors that might influence one-carbon metabolism

    • Standardize tissue collection and processing protocols

  • Cross-validation with multiple antibodies/methods:

    • Use antibodies targeting different epitopes

    • Complement antibody-based detection with mass spectrometry

    • Implement CRISPR-based endogenous tagging to avoid overexpression artifacts

A comprehensive approach employing multiple detection methods was demonstrated in AKI research, where decreased AGXT2 expression was confirmed by both qPCR and immunohistochemistry in a rat model of acute kidney injury .

How can researchers optimize immunohistochemistry protocols for AGXT2 detection in different tissue types?

Optimizing immunohistochemistry protocols for AGXT2 detection requires careful consideration of several technical factors:

  • Tissue fixation and antigen retrieval:

    • For AGXT2 detection in kidney tissues, acetone-methanol (1:1) fixation at 4°C for 10 minutes has been successful

    • Heat-induced epitope retrieval may be necessary for formalin-fixed tissues

    • Different tissue types may require optimization of retrieval buffers (citrate vs. EDTA-based)

  • Blocking and antibody incubation conditions:

    • Protein blocking solutions (e.g., Dako Protein Blocking solution) applied for 20 minutes at room temperature effectively reduce background

    • Primary antibody incubation at 1:100 dilution for 2 hours at 37°C has shown good results for FLAG-tagged AGXT2

    • Secondary antibody incubation at 1:250 dilution for 1 hour at room temperature

  • Co-localization strategies:

    • For vascular studies, co-staining with endothelial markers (CD31) helps identify AGXT2 expression in endothelial cells

    • Mitochondrial markers can confirm proper subcellular localization

    • Nuclear counterstaining with DAPI provides structural context

  • Signal detection and amplification:

    • For weakly expressed AGXT2 in certain tissues, tyramide signal amplification may improve detection

    • Fluorescence detection allows for multiple co-localization studies

    • For chromogenic detection, DAB substrate with hematoxylin counterstaining provides good contrast

  • Tissue-specific considerations:

    • Kidney: AGXT2 is predominantly expressed in renal tubules, particularly proximal tubules

    • Liver: Parenchymal expression pattern requires less stringent antigen retrieval than kidney

    • Vascular tissue: Requires careful blocking of endogenous peroxidases and biotins

A standardized approach for AGXT2 detection in kidney tissue includes fixation, protein blocking, primary antibody incubation (2h, 37°C), washing, secondary antibody incubation (1h, RT), and final visualization with appropriate detection systems .

What are the critical considerations when using AGXT2 antibodies for analyzing transgenic models?

When analyzing transgenic AGXT2 models using antibodies, researchers must address several critical considerations:

  • Distinguishing endogenous from transgenic AGXT2:

    • Use species-specific antibodies when the transgene is from a different species (e.g., human AGXT2 in mice)

    • Employ epitope tag-specific antibodies (e.g., anti-FLAG) when tagged constructs are used

    • Design PCR primers that specifically amplify transgenic versus endogenous transcripts:

      • Human transgenic AGXT2 forward: 5′-GTTGGCAGAGGCAGCATT

      • Human transgenic AGXT2 reverse: 5′-GTCGTCATCCTTGTAATCCTTAGC

  • Controlling for insertion effects:

    • Analyze multiple independent transgenic lines to rule out position effects

    • Assess expression of neighboring genes that might be affected by transgene insertion

    • Compare phenotypes across different expression levels of the transgene

  • Accounting for compensatory mechanisms:

    • Monitor expression of related metabolic enzymes (DDAH1, DDAH2) that might be regulated in response to AGXT2 overexpression

    • Use appropriate housekeeping genes for normalization (e.g., HPRT has been validated)

    • Assess both local (tissue) and systemic (plasma) changes in AGXT2 substrates and products

  • Phenotypic validation:

    • Measure established AGXT2 metabolic markers:

      • Plasma ADMA levels (should decrease with AGXT2 overexpression)

      • ADGV levels (should increase with AGXT2 overexpression)

    • Functional assessments:

      • For vascular studies: endothelial function, pulse pressure, aortic remodeling

      • For renal studies: creatinine, urea nitrogen, and histopathological changes

In one study, transgenic mice showed a 15% decrease in systemic ADMA levels compared to wild type animals, while plasma levels of ADGV were six times higher, confirming functional overexpression of AGXT2 .

How can researchers effectively quantify changes in AGXT2 expression during pathological conditions?

Effective quantification of AGXT2 expression changes during pathological conditions requires a multi-method approach:

  • Transcript level quantification:

    • Implement absolute qPCR quantification using standard curves

    • Select appropriate reference genes that remain stable during the pathological condition

    • For AKI research, significant decreases in AGXT2 mRNA expression have been observed in rat models

  • Protein level assessment:

    • Western blot analysis with densitometric quantification

    • Implement tissue microarrays for high-throughput IHC analysis across multiple samples

    • Consider proteomic approaches for unbiased quantification

  • Functional measurements:

    • Quantify substrate/product ratios (ADMA/ADGV) as functional indicators of AGXT2 activity

    • Measure enzymatic activity in tissue homogenates

    • Assess downstream effects on NO production in relevant tissues

  • Temporal considerations:

    • Design time-course experiments to capture dynamic changes during disease progression

    • Include pre-symptomatic time points to identify early biomarker potential

    • Correlate AGXT2 expression changes with clinical/pathological parameters

  • Statistical analysis and reporting:

    • Use appropriate statistical methods for comparison (e.g., t-tests, ANOVA with post-hoc tests)

    • Report fold-changes with confidence intervals

    • Consider multivariate analysis to identify correlations with other disease markers

A comprehensive approach was demonstrated in AKI research, where both qPCR and immunohistochemistry confirmed decreased AGXT2 expression. The study revealed significant differences in serum biomarkers between control and AKI groups, as shown in this representative data:

Table 1: Biochemical parameters in control vs. AKI rat models

ParameterControl GroupAKI GroupP-value
Serum creatinineNormalIncreased<0.001
Urea nitrogenNormalIncreased<0.001
AGXT2 mRNA expressionReferenceDecreased<0.001

This integrated approach provided strong evidence that AGXT2 downregulation may play a role in AKI pathogenesis .

What are the optimal experimental conditions for using AGXT2 antibodies in Western blot applications?

For optimal Western blot detection of AGXT2, researchers should follow these experimentally validated conditions:

  • Sample preparation:

    • For tissue samples: Homogenize in RIPA buffer containing protease inhibitors

    • For mitochondrial enrichment: Use established mitochondrial isolation protocols

    • Include reducing agents in loading buffer to break potential disulfide bonds

  • Electrophoresis conditions:

    • Use 10-12% SDS-PAGE gels for optimal resolution of AGXT2 (~52 kDa)

    • Load appropriate protein amounts (typically 20-50 μg total protein)

    • Include molecular weight markers covering the 40-60 kDa range

  • Transfer parameters:

    • Semi-dry or wet transfer systems are both suitable

    • Verify transfer efficiency using Ponceau S staining

    • For mitochondrial proteins, consider extended transfer times

  • Blocking and antibody incubation:

    • Block membranes in 5% milk for 1 hour at 37°C

    • For FLAG-tagged AGXT2: Incubate with 1:500 mouse monoclonal anti-FLAG antibody overnight at 4°C

    • For native AGXT2: Optimize primary antibody dilution (typically 1:500-1:2000)

    • Secondary antibody incubation: 1:10,000 HRP-conjugated antibody for 2 hours at room temperature

  • Detection and visualization:

    • Enhanced chemiluminescence systems provide good sensitivity (e.g., Roche Lumi-Light Western Blotting Substrate)

    • Exposure times may need optimization depending on expression levels

    • For quantification, ensure signals are within linear detection range

  • Controls and validation:

    • Include positive control (kidney or liver lysate)

    • Include recombinant AGXT2 or AGXT2-overexpressing cell lysate as reference

    • For FLAG-tagged AGXT2, mouse monoclonal anti-FLAG antibody (Sigma-Aldrich, Catalog #F3165) has shown good specificity

Following these optimized conditions should yield a single, specific band at approximately 52 kDa, representing mature AGXT2 protein.

How can researchers effectively use AGXT2 antibodies to investigate its role in cardiovascular disease models?

To effectively investigate AGXT2's role in cardiovascular disease using antibodies, researchers should implement these approaches:

  • Vascular tissue analysis:

    • Immunohistochemistry of aortic sections to assess AGXT2 expression changes

    • Co-staining with endothelial markers (CD31) to localize AGXT2 in endothelial cells

    • Quantitative analysis of expression in different vascular layers (intima, media, adventitia)

  • Endothelial function studies:

    • Primary aortic cell isolation and culture for in vitro experiments

    • Antibody-based detection of AGXT2 in endothelial cells using immunofluorescence

    • Correlation of AGXT2 expression with markers of endothelial dysfunction

  • Intervention models:

    • AGXT2 overexpression using viral vectors or transgenic approaches

    • Assessment of vascular remodeling in response to AGXT2 modulation

    • Monitoring of pulse pressure as a functional readout of vascular health

  • Molecular mechanism investigation:

    • Co-immunoprecipitation studies to identify AGXT2 interaction partners in vascular tissues

    • Chromatin immunoprecipitation to investigate transcriptional regulation

    • Proximity ligation assays to detect protein-protein interactions in situ

  • Translational applications:

    • Correlation of AGXT2 levels with established cardiovascular biomarkers

    • Stratification of patient samples based on AGXT2 expression patterns

    • Development of predictive models incorporating AGXT2 expression data

This approach has been validated in studies demonstrating that AGXT2 overexpression protects from endothelial dysfunction and adverse aortic remodeling, particularly in the setting of DDAH1 deficiency . The protective effects were associated with lowered plasma ADMA levels and increased ADGV production, confirming AGXT2's functional role in vascular health .

What methodological approaches enable accurate quantification of AGXT2 activity in tissue samples?

Accurate quantification of AGXT2 activity in tissue samples requires specialized methodological approaches:

  • Direct enzyme activity assays:

    • Measurement of transamination reactions using purified mitochondrial fractions

    • Spectrophotometric detection of co-substrates or products

    • Coupling with secondary enzymatic reactions for amplified detection

  • Substrate-product ratio determination:

    • Liquid chromatography/mass spectrometry (LC/MS) quantification of ADMA and ADGV levels

    • Calculation of ADGV/ADMA ratio as an index of AGXT2 activity

    • Analysis in both tissue lysates and plasma samples for comprehensive assessment

  • Isotope-labeled substrate tracing:

    • Use of stable isotope-labeled ADMA to track conversion to ADGV

    • Time-course analysis to determine reaction kinetics

    • Comparison across different tissues to map activity distribution

  • In situ activity visualization:

    • Development of activity-based probes for fluorescence microscopy

    • Correlation with protein expression patterns by immunohistochemistry

    • Co-localization with mitochondrial markers to confirm subcellular activity

  • Correlation with physiological outcomes:

    • Endothelial NO production measurements as a functional readout

    • Vascular reactivity studies in isolated vessel preparations

    • Blood pressure measurements in animal models with modified AGXT2 expression

The measurement of ADMA in plasma or tissue lysates by LC/MS has been established as a reliable approach for inferring AGXT2 activity . In transgenic mice overexpressing AGXT2, plasma ADMA levels were decreased by 15% compared to wild-type animals, while ADGV levels were six times higher, providing clear evidence of enhanced AGXT2 activity .

How should researchers design experiments to investigate AGXT2's role in acute kidney injury?

Based on recent research identifying AGXT2 as an important biomarker for acute kidney injury (AKI) , the following experimental design principles should be applied:

  • Model selection and validation:

    • Implement established AKI models (ischemia-reperfusion, nephrotoxic agents)

    • Confirm AKI development through standard biomarkers (serum creatinine, urea nitrogen)

    • Histopathological verification of renal damage, particularly in proximal tubules

  • Temporal expression analysis:

    • Design time-course experiments capturing pre-injury, acute, and recovery phases

    • Implement qPCR analysis using validated primers for AGXT2 expression quantification

    • Correlate expression changes with progression of kidney injury

  • Protein localization studies:

    • Apply immunohistochemistry to localize AGXT2 in kidney structures

    • Focus on renal tubules where AGXT2 is predominantly expressed

    • Use standardized scoring systems to quantify expression changes

  • Functional studies:

    • Modulate AGXT2 expression (knockdown/overexpression) to assess impact on AKI severity

    • Measure ADMA/ADGV ratios in kidney tissue and plasma during AKI progression

    • Evaluate nitric oxide production as a downstream effector of AGXT2 activity

  • Translational relevance:

    • Design protocols for analysis of human biopsy samples

    • Develop non-invasive methods to assess AGXT2 activity in patients

    • Correlate findings with clinical outcomes in AKI patients

This approach is supported by research showing significant decreases in AGXT2 expression in AKI. In rat models, histopathological examination revealed "significant cytoplasmic swelling and nuclear cleavage of tubular epithelial cells, and renal tubular cell extranuclear changes, mainly in the proximal tubules," coinciding with decreased AGXT2 expression . Both mRNA expression and protein levels of AGXT2 were significantly reduced in AKI rats compared to controls, suggesting a potential role in disease pathogenesis .

What are common sources of non-specific binding with AGXT2 antibodies and how can they be mitigated?

When working with AGXT2 antibodies, researchers may encounter several sources of non-specific binding. These issues and their mitigations include:

  • Cross-reactivity with related aminotransferases:

    • Problem: AGXT2 shares homology with other aminotransferases

    • Mitigation: Use antibodies raised against unique epitopes of AGXT2

    • Validation: Test antibodies on tissues from AGXT2 knockout models

  • High background in mitochondria-rich tissues:

    • Problem: Non-specific binding to abundant mitochondrial proteins

    • Mitigation: Implement more stringent blocking (5% BSA or 5% milk for 1-2 hours)

    • Validation: Include competing peptides to confirm specificity

  • Endogenous biotin interference in IHC/IF:

    • Problem: Biotin-rich tissues can cause high background with biotin-based detection systems

    • Mitigation: Use biotin-blocking steps or non-biotin detection systems

    • Validation: Include biotin-blocking controls in experimental design

  • Fixation artifacts:

    • Problem: Overfixation can mask epitopes and cause non-specific binding

    • Mitigation: Optimize fixation protocols (acetone-methanol 1:1 for 10 min at 4°C has shown good results)

    • Validation: Compare different fixation methods in parallel

  • Secondary antibody cross-reactivity:

    • Problem: Secondary antibodies may bind non-specifically to endogenous immunoglobulins

    • Mitigation: Use secondary antibodies pre-adsorbed against the species being studied

    • Validation: Include secondary-only controls

For Western blot applications specifically, blocking membranes in 5% milk for 1 hour at 37°C followed by overnight primary antibody incubation at 4°C has been shown to minimize non-specific binding . For immunohistochemistry, pre-incubation with Dako Protein Blocking solution for 20 minutes at room temperature effectively reduces background staining .

How can researchers address challenges in detecting endogenous AGXT2 in different experimental systems?

Detecting endogenous AGXT2 presents several challenges that researchers can address through these methodological approaches:

  • Low expression level detection:

    • Challenge: AGXT2 may be expressed at low levels in certain tissues

    • Solution: Implement signal amplification techniques (tyramide signal amplification for IHC)

    • Approach: Enrich mitochondrial fractions before Western blot analysis

  • Tissue-specific optimization:

    • Challenge: Different tissues require different processing for optimal detection

    • Solution: Develop tissue-specific protocols (e.g., kidney vs. liver)

    • Approach: For kidney samples, focus on renal tubules where AGXT2 expression is highest

  • Antibody selection for specific applications:

    • Challenge: Not all antibodies work equally well across applications

    • Solution: Validate antibodies specifically for each application (WB, IHC, IF, IP)

    • Approach: For IHC, rabbit polyclonal antibodies have shown good results in kidney tissue

  • Species cross-reactivity issues:

    • Challenge: Antibodies may not cross-react between species

    • Solution: Verify species reactivity or use species-specific antibodies

    • Approach: Some commercial antibodies are verified for both human and pig samples

  • Subcellular localization confirmation:

    • Challenge: Confirming mitochondrial localization in tissue sections

    • Solution: Co-staining with established mitochondrial markers

    • Approach: Use confocal microscopy for precise localization studies

For Western blot applications, enriching mitochondrial fractions significantly improves detection sensitivity. One validated approach involves isolating mitochondria using established protocols, followed by FLAG affinity chromatography when working with tagged constructs . For immunohistochemistry of kidney samples, focusing on renal tubules where AGXT2 is predominantly expressed provides optimal detection sensitivity .

What quality control measures should researchers implement when working with newly procured AGXT2 antibodies?

When working with newly procured AGXT2 antibodies, researchers should implement these essential quality control measures:

  • Initial validation:

    • Verify antibody information (host species, clonality, immunogen details)

    • Check literature for previous validation of the same antibody clone/lot

    • Review manufacturer's validation data critically

  • Application-specific testing:

    • Western blot: Confirm single band at expected molecular weight (~52 kDa)

    • IHC/IF: Verify expected subcellular localization (mitochondrial pattern)

    • IP: Confirm ability to immunoprecipitate AGXT2 from lysates

  • Positive and negative controls:

    • Positive tissue controls: Kidney and liver samples (high AGXT2 expression)

    • Negative controls: Samples from AGXT2 knockout models

    • Neutralization controls: Pre-incubation with immunizing peptide

  • Reproducibility assessment:

    • Test multiple lots of the same antibody if available

    • Evaluate consistency across different experimental runs

    • Compare results across different detection methods

  • Cross-validation with orthogonal methods:

    • Validate expression findings with qPCR

    • Confirm protein detection with mass spectrometry

    • Correlate antibody-based detection with enzyme activity assays

  • Systematic reporting:

    • Document all validation steps performed

    • Record antibody details (manufacturer, catalog number, lot number)

    • Share validation data when publishing results

A comprehensive approach for AGXT2 antibody validation was demonstrated in studies where both recombinant protein expression systems and tissue analyses were employed . For example, FLAG-tagged AGXT2 was detected with mouse monoclonal anti-FLAG antibody (Sigma-Aldrich, Catalog #F3165) at 1:500 dilution, which showed specific detection of the target protein . Similarly, for immunofluorescence applications, rabbit polyclonal anti-FLAG antibodies (Sigma-Aldrich, Catalog #7425) at 1:100 dilution produced specific staining of FLAG-tagged AGXT2 .

How can AGXT2 antibodies be utilized in multi-omics research approaches?

AGXT2 antibodies can be effectively integrated into multi-omics research through these approaches:

  • Integration with proteomics:

    • Immunoprecipitation followed by mass spectrometry to identify AGXT2 interaction partners

    • Antibody-based enrichment of mitochondrial proteins for targeted proteomics

    • Combining AGXT2 antibody-based detection with global proteome profiling

  • Proteogenomic applications:

    • Correlation of protein expression (antibody-based) with transcriptomic data

    • Integration with genotyping information for SNPs affecting AGXT2 function

    • Analysis of post-transcriptional regulation mechanisms

  • Spatial multi-omics:

    • Antibody-based spatial profiling of AGXT2 in tissue sections

    • Correlation with spatial transcriptomics data from adjacent sections

    • Mapping of metabolic gradients in relation to AGXT2 expression patterns

  • Single-cell applications:

    • Combined single-cell RNA-seq with antibody-based protein detection

    • Analysis of cell-specific AGXT2 expression in heterogeneous tissues

    • Correlation with cell-type specific metabolic profiles

  • Clinical multi-omics:

    • Antibody-based tissue microarrays correlated with patient -omics data

    • Development of multi-parameter predictive models incorporating AGXT2

    • Identification of patient subgroups based on integrated analyses

This approach has been partially demonstrated in AKI research, where transcriptome analysis using RNA sequencing data from kidney biopsy specimens identified AGXT2 as one of the top three genes with the most connected nodes based on protein-protein interaction network analysis . The downregulation of AGXT2 was subsequently confirmed at both mRNA and protein levels using qPCR and immunohistochemistry, respectively .

What novel research directions are emerging for investigating AGXT2's role in metabolism and disease?

Several novel research directions are emerging for investigating AGXT2's role in metabolism and disease:

  • Expanded role in vascular biology:

    • Investigation of AGXT2 as a therapeutic target for endothelial dysfunction

    • Exploration of pharmacological approaches to upregulate AGXT2 as an alternative to DDAH-based interventions

    • Development of AGXT2 activators for treatment of ADMA-related vascular pathologies

  • Metabolic disease connections:

    • Investigation of AGXT2's role in metabolic syndrome

    • Exploration of connections to insulin resistance and glucose metabolism

    • Analysis of AGXT2 polymorphisms associated with metabolic disease risk

  • Kidney disease biomarkers:

    • Development of AGXT2-based biomarkers for early detection of AKI

    • Longitudinal studies correlating AGXT2 expression with kidney disease progression

    • Integration of AGXT2 into multi-marker panels for kidney injury assessment

  • Mitochondrial biology intersections:

    • Investigation of AGXT2's role in mitochondrial stress responses

    • Analysis of interactions between AGXT2 and mitochondrial quality control mechanisms

    • Exploration of connections to mitochondrial metabolism beyond aminotransferase activity

  • Novel substrate exploration:

    • Comprehensive metabolomic analysis to identify additional AGXT2 substrates

    • Investigation of D-amino acid metabolism in mammalian systems

    • Analysis of AGXT2's role in detoxifying non-canonical amino acids

Recent research has identified AGXT2 as one of the top three genes (along with SHMT1 and ACO2) significantly associated with acute kidney injury through weighted gene co-expression network analysis (WGCNA) . This finding opens new avenues for exploring AGXT2's role in kidney pathophysiology and its potential as a diagnostic biomarker or therapeutic target. Additionally, the demonstrated protection from ADMA-induced endothelial dysfunction through AGXT2 overexpression highlights its potential therapeutic value in cardiovascular disease .

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