AGPAT1 Antibody

What is AGPAT1 and what cellular processes does it participate in?

AGPAT1 (1-acylglycerol-3-phosphate O-acyltransferase 1) is an enzyme responsible for the conversion of lysophosphatidic acid into phosphatidic acid, serving as a key intermediate in phospholipid biosynthesis pathways . This enzyme plays critical roles in lipid metabolism with growing interest in its function as a regulator of immune responses . AGPAT1 is ubiquitously expressed across tissues, unlike its homolog AGPAT2 which shows more restricted tissue distribution . Recent studies have demonstrated AGPAT1's significance in multiple physiological processes including glucose metabolism, reproductive development, and neurological function .

Which experimental applications are validated for AGPAT1 antibodies?

AGPAT1 antibodies have been validated for multiple experimental applications including Western Blot (WB), Immunoprecipitation (IP), Immunohistochemistry (IHC), and ELISA . For immunoprecipitation, positive detection has been confirmed in Raji cells and mouse skeletal muscle tissue . In immunohistochemistry, successful detection has been observed in human skeletal muscle tissue and human testis tissue . Published literature confirms the utility of AGPAT1 antibodies in Western Blot applications across multiple studies . When selecting an antibody for your research, consider the specific validated reactivity with human, mouse, or rat samples depending on your experimental model .

What dilutions are recommended for different AGPAT1 antibody applications?

The optimal dilution of AGPAT1 antibody varies by application technique and specific antibody product. For recommended standard dilutions:

It is strongly recommended to empirically titrate the antibody in each specific testing system to obtain optimal results, as performance may be sample-dependent . For paraffin-embedded tissue sections in IHC applications, a dilution of 1:100 has been successfully used for human kidney and placenta samples .

What antigen retrieval methods are recommended for IHC with AGPAT1 antibodies?

For optimal immunohistochemical detection of AGPAT1, TE buffer pH 9.0 is the suggested antigen retrieval method . As an alternative approach, antigen retrieval may also be performed with citrate buffer pH 6.0 . The choice between these methods may depend on your specific tissue type and fixation protocol. In published research evaluating AGPAT1 as a biomarker in PSC-UC versus UC, researchers successfully performed IHC using a validated AGPAT1 antibody (HPA073355, Atlas Antibodies AB) on formalin-fixed paraffin-embedded (FFPE) colonic specimens . The staining pattern was generally cytoplasmic in both epithelial and inflammatory cells, with surface epithelial cells displaying more intense staining compared to crypt epithelial cells .

How can AGPAT1 expression be quantified in tissue samples for biomarker research?

For quantitative assessment of AGPAT1 expression, multiple complementary approaches are recommended. In proteomic analyses, LC-MS/MS methods can be employed following multienzyme digestion filter-aided sample preparation (FASP) . This approach allows for high-resolution protein identification and relative quantification, as demonstrated in studies comparing PSC-UC and UC patient samples . For immunohistochemical quantification, automated image analysis software provides objective assessment of staining intensity. In published research, QuPath (version 0.2.3) has been successfully implemented to evaluate AGPAT1 IHC intensity in colonic biopsies .

The protocol involves:

• Selection of representative biopsies from each patient

• Measurement of DAB-stained area within the whole biopsy using moderate resolution, Gaussian prefilter, and smoothing sigma of 2

• Application of three different thresholds for positive staining intensity: weak (0.05–0.14), moderate (0.15–0.29), and strong (0.30)

• Calculation of a total intensity score using the algorithm: (×1% weakly stained area) + (×2% moderately stained area) + (×3% strongly stained area)

This methodological approach provides a reproducible quantification system that has successfully mirrored mass spectrometry findings in comparative studies .

What are the implications of AGPAT1 as a biomarker in inflammatory bowel disease?

AGPAT1 has emerged as a promising biomarker for discriminating between "classical" ulcerative colitis (UC) and primary sclerosing cholangitis-associated UC (PSC-UC) . In a two-step validation approach using proteomic analysis followed by independent cohort verification, AGPAT1 showed significantly higher expression in PSC-UC compared to UC patients . This differential expression was confirmed at both the protein level through mass spectrometry and at the tissue level via immunohistochemistry with automated quantification .

The statistical validation of AGPAT1 as a biomarker is summarized in the following data:

Of note, AGPAT1 was the only biomarker among five candidates that was successfully validated in the replication cohort, suggesting its robust discriminatory potential . Furthermore, meta-analysis of combined proteomic datasets identified AGPAT1 as having the lowest P-value (3.6e-06) among 6,121 proteins analyzed, with significance retained after adjusting for multiple testing and controlling for age and thiopurine use . This evidence positions AGPAT1 as a potential clinically useful biomarker that could be incorporated into routine pathology through IHC-based evaluation.

What phenotypes are observed in AGPAT1-deficient animal models?

AGPAT1-deficient mouse models (Agpat1-/-) demonstrate widespread physiological disturbances across multiple organ systems, highlighting the enzyme's essential role in normal development and function . Key phenotypic manifestations include:

• Metabolic abnormalities:

  • Reduced survival before weaning

  • Low body weight

  • Low plasma glucose levels (independent of plasma insulin and glucagon levels)

  • Reduced percentage of body fat without generalized lipodystrophy

  • Decreased hepatic mRNA expression of Igf-1 and Foxo1, suggesting impaired gluconeogenesis

• Reproductive abnormalities:

  • In male mice: impaired sperm development with late meiotic arrest near the onset of round spermatid production, accompanied by elevated gonadotropins

  • In female mice: oligoanovulation with retained responsiveness to gonadotropins

• Neurological abnormalities:

  • Abnormal hippocampal neuron development

  • Development of audiogenic seizures

These phenotypes result from disrupted phospholipid homeostasis and suggest that AGPAT1 serves critical functions in the physiology of multiple organ systems despite having seemingly redundant enzymatic activity with other AGPAT isoforms . The Agpat1-/- genotype also shows reduced viability, with breeding between Agpat1+/- mice yielding progeny ratios of 1:2:0.5 (wildtype:heterozygous:knockout) instead of the expected 1:2:1 Mendelian ratio (P < 0.001) .

How can researchers distinguish between different AGPAT isoforms in experimental systems?

Distinguishing between AGPAT isoforms requires a multifaceted approach combining isoform-specific detection methods and knowledge of tissue expression patterns. While AGPAT1 and AGPAT2 share similar in vitro substrate specificities as recombinant proteins, their tissue expression patterns differ significantly . AGPAT1 shows ubiquitous expression across tissues, whereas AGPAT2 exhibits more restricted tissue distribution .

In mouse tissues, expression analysis reveals that Agpat1 is expressed at lower levels than Agpat2 in metabolically active tissues: approximately 14-fold lower in liver, 1.5-fold lower in epididymal fat, and 5.8-fold lower in brown fat . These differential expression patterns provide one approach to distinguishing isoforms in experimental systems.

For specific detection of AGPAT1:

• Use validated isoform-specific antibodies that have been tested for cross-reactivity with other AGPAT family members

• Employ quantitative PCR with primers specific to AGPAT1 transcripts

• Consider tissue context when interpreting results, as expression levels vary significantly between tissues

• In knockout or knockdown studies, verify the specific targeting of AGPAT1 rather than other isoforms

The observed phenotypes in Agpat1-/- mice despite the presence of other AGPAT isoforms suggests non-redundant functions that may relate to subcellular localization, tissue-specific regulation, or unique protein-protein interactions beyond enzymatic activity .

What controls should be included when using AGPAT1 antibodies for research?

When designing experiments with AGPAT1 antibodies, rigorous controls are essential for result interpretation and validation:

• Positive controls:

  • For Western blot: K562 whole cell lysate has been validated to show the expected 32 kDa band size

  • For IP: Raji cells and mouse skeletal muscle tissue have shown positive detection

  • For IHC: Human skeletal muscle tissue and human testis tissue are recommended positive control tissues

• Negative controls:

  • Isotype-matched irrelevant antibody to evaluate non-specific binding

  • Samples from AGPAT1 knockout models where available

  • Primary antibody omission to assess secondary antibody specificity

• Specificity controls:

  • Peptide competition assays using the immunogen peptide to confirm binding specificity

  • Comparison with other validated AGPAT1 antibodies targeting different epitopes

  • Correlation of protein detection with mRNA expression data

• Loading/technique controls:

  • For WB: Housekeeping protein controls (β-actin, GAPDH)

  • For IHC: Internal tissue elements with known expression patterns

  • For quantitative analyses: Standard curves with recombinant protein when applicable

When interpreting results, verification of the expected molecular weight (32 kDa) for AGPAT1 protein is critical . Additionally, researchers should be aware that in IHC applications, AGPAT1 shows differential staining intensity between surface epithelial cells and crypt epithelial cells, which could affect quantification strategies .

How can proteomic approaches be optimized for AGPAT1 detection in FFPE tissues?

Detection of AGPAT1 in formalin-fixed paraffin-embedded (FFPE) tissues presents unique challenges due to protein cross-linking and potential epitope masking. Successful proteomic analysis of AGPAT1 in FFPE samples has been achieved using the following optimization strategies:

• Sample preparation:

  • Implement multienzyme digestion filter-aided sample preparation (FASP) method

  • Utilize consecutive digestion with endoproteinase LysC followed by trypsin to improve protein recovery

  • Quantify total protein and peptide yield using the tryptophan fluorescence (WF) assay to ensure sufficient material for analysis

• LC-MS/MS analysis:

  • Separate peptides on reverse phase C18 column using an extended (95-minute) acetonitrile gradient to improve resolution

  • Acquire spectra using high-resolution instruments (e.g., QExactive)

  • Process data with appropriate software (e.g., MaxQuant) for protein identification and quantification

  • Apply the "total protein approach" using raw spectral intensities for protein quantification

• Validation strategies:

  • Confirm MS findings through orthogonal methods like immunohistochemistry

  • Use step-wise discovery and validation cohorts to reduce false positive findings

  • Consider meta-analysis of combined datasets to strengthen confidence in findings

This technical approach has proven successful in biomarker studies using archived FFPE samples, with AGPAT1 specifically identified as a discriminatory protein between PSC-UC and UC patient samples . The method enables high-resolution proteome analysis of archived biological samples and has been validated as technically and practically feasible in retrospective IBD research .

What are the key considerations for interpreting AGPAT1 knockout studies in relation to human disease?

When interpreting AGPAT1 knockout studies in animal models and extrapolating findings to human disease contexts, several critical considerations must be addressed:

These considerations highlight the complexity of interpreting knockout studies and the importance of comprehensive, multi-modal approaches when translating findings to human disease contexts.

How is AGPAT1 implicated in cancer research beyond its role as a biomarker?

Beyond its utility as a diagnostic biomarker, AGPAT1 has emerging significance in cancer research, particularly in colorectal cancer. AGPAT1 has been identified as a negative prognostic marker for colorectal cancer outcomes . This finding has been confirmed through prognostic analysis of data from The Cancer Genome Atlas (TCGA), as referenced in the Human Protein Atlas (https://www.proteinatlas.org/ENSG00000204310-AGPAT1/pathology/colorectal+cancer)[2].

The mechanistic link between AGPAT1 and cancer progression likely involves its role in lipid metabolism. Growing evidence suggests that lipid metabolic pathways are critically important for colorectal cancer development . AGPAT1's enzymatic function in converting lysophosphatidic acid to phosphatidic acid positions it at a crucial junction in phospholipid synthesis pathways that may influence:

• Cell membrane composition and fluidity affecting receptor signaling

• Production of lipid second messengers involved in proliferation and survival signaling

• Energy metabolism adaptations characteristic of cancer cells

• Inflammatory microenvironment regulation through lipid mediators

Research examining AGPAT1 expression patterns in tumor versus normal tissue, correlations with clinical outcomes, and functional studies manipulating AGPAT1 levels in cancer models represent promising directions for understanding its role in oncogenesis and potential therapeutic targeting.

What methodological approaches can resolve contradictions in AGPAT1 functional studies?

Resolving contradictions in AGPAT1 functional studies requires integrated methodological approaches that address the complexity of lipid metabolism regulation and tissue-specific effects:

• Comprehensive isoform analysis:

  • Simultaneous profiling of all AGPAT family members using quantitative PCR and protein-level measurements

  • Assessment of compensatory mechanisms among isoforms in different experimental conditions

  • Consideration of splice variants and post-translational modifications affecting function

• Cell type-specific investigations:

  • Single-cell analysis techniques to resolve heterogeneous cell populations within tissues

  • Cell type-specific conditional knockout models to distinguish autonomous versus non-autonomous effects

  • Co-culture systems to evaluate intercellular communication and paracrine effects

• Combined -omics approaches:

  • Integration of transcriptomics, proteomics, and lipidomics data to build comprehensive pathway models

  • Temporal analyses capturing dynamic changes in response to perturbation

  • Computational modeling to predict systemic effects of AGPAT1 modulation

• Rigorous technical standardization:

  • Standardized antibody validation across multiple detection methods

  • Consistent sample preparation protocols for cross-study comparisons

  • Reporting of detailed methodological parameters including antibody dilutions, antigen retrieval methods, and quantification algorithms

• Physiological context preservation:

  • Studies in primary cells and tissues rather than immortalized cell lines when possible

  • Consideration of microenvironmental factors including substrate availability and hormonal influences

  • In vivo validation of in vitro findings through targeted genetic manipulations

Implementing these approaches can help reconcile apparently contradictory findings by revealing context-dependent functions and regulatory mechanisms that may not be apparent in more reductionist experimental systems.

What are the technical challenges in developing specific detection methods for AGPAT1 versus other AGPAT isoforms?

Developing highly specific detection methods for AGPAT1 versus other AGPAT isoforms presents several technical challenges that researchers must address:

• Sequence homology issues:

  • Human AGPAT1 and AGPAT2 are close homologues with similar in vitro substrate specificities

  • Antibody development must target unique epitopes that distinguish between isoforms

  • Epitope mapping and cross-reactivity testing against all AGPAT family members is essential

• Transcript detection challenges:

  • Design of isoform-specific primers must account for potential splice variants

  • Quantitative PCR requires rigorous validation of primer specificity across all isoforms

  • RNA-seq analysis needs sufficient read depth and appropriate bioinformatic pipelines to discriminate between similar transcripts

• Protein detection optimization:

  • Western blot conditions (antibody concentration, blocking agents, incubation times) must be optimized for specificity

  • For IHC applications, antigen retrieval methods significantly impact epitope accessibility and specificity

  • Recommended protocols include TE buffer pH 9.0 or alternatively citrate buffer pH 6.0

• Subcellular localization considerations:

  • AGPAT isoforms may have distinct subcellular localizations requiring appropriate fixation and permeabilization protocols

  • Double-labeling with organelle markers can help resolve localization patterns

  • Super-resolution microscopy may be needed to distinguish closely associated isoforms

• Activity-based detection:

  • Enzymatic activity assays must be designed with isoform-specific substrates or conditions

  • Inhibitor studies require compounds with demonstrated selectivity between isoforms

  • In situ activity detection presents particular challenges due to the presence of multiple isoforms

Addressing these challenges requires a multi-modal approach combining genomic, transcriptomic, proteomic, and functional assays with rigorous validation across different experimental systems.

How might AGPAT1-targeted therapies be developed based on current understanding?

Development of AGPAT1-targeted therapies represents an emerging frontier based on the enzyme's role in multiple physiological processes and disease states. Strategic approaches for therapeutic development include:

• Inhibitor design strategies:

  • Structure-based design targeting the catalytic site of AGPAT1

  • Allosteric modulators affecting enzyme conformation or substrate binding

  • Isoform-selective inhibitors that exploit structural differences between AGPAT family members

• Disease-specific targeting approaches:

  • For inflammatory conditions: Modulation of AGPAT1 activity to alter lipid mediator production in inflammatory bowel disease contexts

  • For metabolic disorders: Tissue-specific targeting of AGPAT1 in metabolically active tissues

  • For cancer: Development of inhibitors affecting phospholipid synthesis in rapidly proliferating cells

• Delivery system considerations:

  • Tissue-specific delivery methods to minimize off-target effects

  • Temporal control of inhibition to avoid developmental complications observed in knockout models

  • Combination approaches with existing therapeutic modalities

• Biomarker-guided therapy:

  • Utilization of AGPAT1 expression levels as a predictive biomarker for treatment response

  • Integration of AGPAT1 status in patient stratification for clinical trials

  • Development of companion diagnostics alongside therapeutic agents

• Genetic therapy approaches:

  • RNA interference strategies for temporary knockdown in specific tissues

  • CRISPR-based gene editing for permanent modification in appropriate contexts

  • Gene therapy approaches for conditions where AGPAT1 deficiency is implicated

The multi-system effects observed in AGPAT1-deficient mice emphasize the importance of carefully designed therapeutic interventions with targeted tissue specificity and appropriate dosing to achieve desired effects while minimizing adverse outcomes.

What are the most promising methodological innovations for studying AGPAT1 regulation and function?

Emerging methodological innovations offer new approaches to understanding AGPAT1 regulation and function with unprecedented precision:

• Advanced imaging techniques:

  • CRISPR-mediated endogenous tagging of AGPAT1 for live-cell imaging

  • Super-resolution microscopy to visualize AGPAT1 subcellular localization and dynamics

  • Correlative light and electron microscopy (CLEM) to relate protein localization to ultrastructural features

• Spatially-resolved -omics:

  • Spatial transcriptomics to map AGPAT1 expression patterns within heterogeneous tissues

  • Imaging mass spectrometry for spatial lipidomics to correlate AGPAT1 activity with lipid distribution

  • Digital spatial profiling combining protein and RNA detection in tissue sections

• Functional genomics approaches:

  • CRISPR screening to identify regulatory networks and functional interactors

  • Precise genome editing to introduce disease-associated variants

  • Single-cell multi-omics to correlate genotype, transcriptome, and phenotype

• Structural biology advances:

  • Cryo-electron microscopy for high-resolution protein structure determination

  • Hydrogen-deuterium exchange mass spectrometry to study protein dynamics

  • Computational modeling of enzyme-substrate interactions and inhibitor binding

• In situ biochemistry:

  • Activity-based protein profiling to measure AGPAT1 enzymatic activity in living systems

  • Proximity labeling methods to identify protein interaction networks

  • Optogenetic and chemogenetic tools for acute manipulation of AGPAT1 function

These methodological innovations promise to reveal new insights into AGPAT1 function beyond its basic enzymatic role, potentially uncovering non-canonical functions and regulatory mechanisms that could explain its essential role in multiple physiological processes despite apparent redundancy with other AGPAT isoforms .

How can interdisciplinary approaches enhance understanding of AGPAT1 biology?

Understanding the complex biology of AGPAT1 requires interdisciplinary collaboration across multiple research domains:

• Biochemistry and structural biology:

  • Detailed characterization of AGPAT1 enzymatic mechanisms

  • Structural determination of protein-substrate and protein-protein interactions

  • Investigation of post-translational modifications affecting enzyme activity

• Cell biology and physiology:

  • Analysis of subcellular localization and trafficking

  • Tissue-specific functions in development and homeostasis

  • Cellular response to AGPAT1 manipulation in different physiological states

• Systems biology and bioinformatics:

  • Network analysis integrating AGPAT1 into broader metabolic and signaling pathways

  • Multi-omics data integration and modeling

  • Prediction of functional consequences for genetic variants

• Clinical research and pathology:

  • Correlation of AGPAT1 expression with disease progression and outcomes

  • Development and validation of AGPAT1 as a biomarker in inflammatory conditions

  • Analysis of tissue-specific expression patterns in health and disease

• Pharmacology and drug discovery:

  • Design and screening of selective AGPAT1 modulators

  • Evaluation of inhibitor efficacy in disease models

  • Assessment of combinatorial approaches targeting multiple aspects of lipid metabolism

Collaborative research frameworks that integrate these diverse disciplines can address complex questions about AGPAT1 function that would be difficult to resolve within a single research domain. The convergence of methodological expertise and conceptual frameworks will be essential for translating basic biological insights into clinically relevant applications.