AGPAT9 Antibody

What is AGPAT9 and how is it relevant to cellular metabolism?

AGPAT9 (1-acylglycerol-3-phosphate O-acyltransferase 9), also known as GPAT3 (glycerol-3-phosphate acyltransferase 3), is a member of the 1-acyl-sn-glycerol-3-phosphate acyltransferase protein family . The human canonical protein has 434 amino acid residues with a molecular mass of approximately 48.7 kDa . AGPAT9 is localized to the endoplasmic reticulum and is widely expressed across diverse tissue types .

AGPAT9 plays a significant role in lipid metabolism pathways and has been identified as being involved in various metabolic processes. The protein was first identified from adipose tissue in 2007 . Importantly, AGPAT9 has emerged as an important focus in cancer research, particularly breast cancer, where it appears to have tumor-suppressive properties .

What applications are most validated for AGPAT9 antibodies?

AGPAT9 antibodies have been validated for multiple research applications including:

• Western Blot (WB): For detecting the ~49 kDa AGPAT9 protein in tissue and cell lysates

• Immunohistochemistry (IHC): Particularly useful for paraffin-embedded tissues with recommendations for HIER pH 6 retrieval

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of native AGPAT9 in biological samples

• Immunocytochemistry (ICC) and Immunofluorescence (IF): For cellular localization studies

For optimal results in IHC-Paraffin applications, antibody dilutions of 1:500 to 1:1000 are typically recommended . When using the antibody for IHC applications, human duodenum tissue shows strong cytoplasmic positivity with a characteristic granular pattern in glandular cells .

How should researchers select appropriate controls when using AGPAT9 antibodies?

When designing experiments with AGPAT9 antibodies, the following control strategy is recommended:

• Positive tissue controls: Use tissues known to express AGPAT9, such as adipose tissue where it was originally identified, or duodenum which shows strong cytoplasmic staining patterns .

• Cellular controls: Consider using MCF-7 breast cancer cells which demonstrate relatively high AGPAT9 expression compared to MDA-MB-231 cells .

• Knockdown/overexpression controls: Generate stable cell lines with either AGPAT9 knockdown (using shRNA) or overexpression (via lentiviral vectors) as described in research protocols . These provide critical validation for antibody specificity.

• Cross-reactivity assessment: Due to the homology between AGPAT family members, peptide competition assays or using tissues from AGPAT9 knockout models would confirm specificity.

• Isotype controls: Include appropriate rabbit IgG controls when using rabbit polyclonal antibodies to control for non-specific binding .

What methodologies are most effective for studying AGPAT9's role in cancer progression?

Research indicates that AGPAT9 has tumor-suppressive properties in breast cancer models. To investigate this function effectively, the following methodological approaches have proven successful:

Cell line models and expression manipulation:

• Establish stable AGPAT9-overexpressing cell lines in highly invasive breast cancer models (e.g., MDA-MB-231) using lentiviral vectors carrying the AGPAT9 gene .

• Create AGPAT9 knockdown models in less invasive cell lines (e.g., MCF-7) using shRNA technology targeting specific AGPAT9 sequences .

Functional assays:

• Proliferation assays:

  • CCK-8 cell proliferation assay

  • Real-time cell proliferation monitoring using the xCELLigence system

  • Colony formation assays

• Migration and invasion assays:

  • Transwell migration assays

  • Matrigel invasion assays (using Matrigel at ~8.95 mg/ml protein concentration)

  • Live-cell imaging with confocal scanner systems

  • Real-time migration/invasion monitoring using the xCELLigence system

• In vivo models:

  • Subcutaneous xenograft models (e.g., MCF7 cells with AGPAT9 knockdown)

  • Lung metastasis models (tail vein injection of MDA-MB-231 cells with AGPAT9 overexpression)

The combination of these approaches has demonstrated that AGPAT9 significantly inhibits breast cancer cell proliferation both in vitro and in vivo, and significantly reduces migration and invasion capabilities .

How can researchers effectively detect changes in AGPAT9 expression and correlate them with functional outcomes?

To effectively measure AGPAT9 expression changes and correlate them with functional outcomes, researchers should employ a multi-faceted approach:

Expression analysis techniques:

• qRT-PCR: Using validated primers such as:

  • Forward: 5'-CACCGTGACCGACCTATTC-3'

  • Reverse: 5'-GCCCAGCGTCTGAGTTTT-3'

• Western blot analysis: Using affinity-purified antibodies specific for human AGPAT9, typically detecting a band at ~49 kDa .

• Functional correlation assays:

  • V-ATPase activity measurement to assess the impact of AGPAT9 on proton pump function

  • Extracellular pH (pHe) and intracellular pH (pHi) measurements

  • MMP-2 and MMP-9 activity assays using commercial kits

• Mechanistic studies:

  • Chromatin immunoprecipitation (ChIP) assays to investigate transcriptional regulation

  • Co-immunoprecipitation to detect protein-protein interactions

Research has shown that AGPAT9 inhibits breast cancer cell proliferation, migration, and invasion partly by suppressing V-ATPase activity, which affects extracellular and intracellular pH. Additionally, AGPAT9 upregulates KLF4 and LASS2 expression, which appears to be part of its tumor-suppressive mechanism .

What are the challenges in studying AGPAT9 protein interactions and how can they be addressed?

Investigating AGPAT9 protein interactions presents several technical challenges that researchers should address through careful experimental design:

Challenges and solutions:

• Membrane protein localization:

  • AGPAT9 localizes to the endoplasmic reticulum, making protein extraction challenging

  • Solution: Use specialized extraction buffers designed for membrane proteins, such as those containing mild detergents that preserve protein structure

• Similar sequence homology with other AGPAT family members:

  • Solution: Use epitope-specific antibodies targeting unique regions of AGPAT9 that differ from other family members

• Detecting protein-protein interactions:

  • For studying interaction with V-ATPase components or other signaling proteins

  • Solution: Proximity ligation assays (PLA) can detect protein interactions in situ with high specificity

• Tracking AGPAT9 in living cells:

  • Solution: Generation of fluorescently tagged AGPAT9 constructs (e.g., GFP-tagged) has been successfully used to visualize subcellular localization

What experimental designs best elucidate AGPAT9's role in chemosensitivity of cancer cells?

Research has indicated that AGPAT9 may play a role in chemosensitivity, particularly in breast cancer. To investigate this function:

Experimental design recommendations:

• Cell line model selection:

  • Use paired drug-sensitive and drug-resistant cell lines (e.g., MCF7 and MCF7/ADR)

  • AGPAT9 expression has been found to be significantly decreased (-240.368-fold) in drug-resistant MCF7/ADR cells compared to drug-sensitive MCF7 cells

• AGPAT9 manipulation strategies:

  • Generate stable AGPAT9-overexpressing cell lines in drug-resistant models using lentiviral vectors

  • Assess IC50 values for various chemotherapeutic agents (e.g., doxorubicin)

• Mechanistic investigation:

  • Subcellular distribution studies of chemotherapeutic agents (e.g., doxorubicin) using confocal fluorescence microscopy

  • Analysis of drug efflux pump expression and function

  • pH gradient measurements across cellular compartments

• Combination therapy assessment:

  • Test whether AGPAT9-modulating compounds can sensitize resistant cells to standard chemotherapeutics

  • Investigate potential synergistic effects through combination index calculations

Research has shown that overexpression of AGPAT9 in MCF7/ADR cells significantly reduced the IC50 value for doxorubicin and altered the subcellular distribution of the drug, leading to enhanced nuclear targeting instead of cytoplasmic sequestration .

What optimization steps are crucial when using AGPAT9 antibodies for different applications?

For optimal results with AGPAT9 antibodies across different applications, researchers should consider the following optimization strategies:

For Western Blotting:

• Sample preparation: Use RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 50 mM Tris, pH 8.0) containing protease inhibitors for cell lysis

• Protein loading: Load 40 μg of total cell lysate proteins for adequate detection

• Recommended dilutions: Start with 1:1000 dilution and adjust as needed

• Blocking: Use 5% non-fat milk containing 0.2% Tween 20

• Detection system: Enhanced chemiluminescence reagents typically provide sufficient sensitivity

For Immunohistochemistry:

• Antigen retrieval: HIER pH 6 retrieval is recommended for paraffin sections

• Antibody dilution: 1:500 - 1:1000 typically yields optimal results

• Detection system: Use appropriate secondary antibodies such as Goat Anti-Rabbit IgG conjugated with biotin, FITC, or HRP depending on the detection method

• Positive control tissues: Human duodenum shows strong cytoplasmic positivity with a granular pattern in glandular cells

For ELISA:

• Sample types: Undiluted body fluids and/or tissue homogenates are appropriate

• Detection range: Consider the theoretical kit detection range for your specific sample type

• Cross-reactivity: Be aware that antibodies may detect native, not recombinant AGPAT9

How can researchers troubleshoot inconsistent results when working with AGPAT9 antibodies?

When encountering inconsistent results with AGPAT9 antibodies, consider the following troubleshooting strategies:

Common issues and solutions:

• Weak or no signal in Western blots:

  • Verify protein extraction efficiency from membrane fractions

  • Increase antibody concentration or extend incubation time

  • Ensure transfer efficiency for membrane proteins

  • Consider using specialized membrane protein extraction buffers

• Multiple bands or unexpected molecular weight:

  • AGPAT9 theoretical molecular weight is 49 kDa, but post-translational modifications may alter migration

  • Cross-reactivity with other AGPAT family members might occur due to sequence homology

  • Verify antibody specificity using AGPAT9 knockdown or overexpression controls

• Variable staining in IHC:

  • Optimize antigen retrieval conditions (HIER pH 6 is recommended)

  • Test multiple antibody dilutions (1:500 - 1:1000 range)

  • Use properly fixed tissues (overfixation can mask epitopes)

  • Include positive control tissues (duodenum shows reliable staining)

• Inconsistent functional study results:

  • Confirm AGPAT9 expression levels by both qRT-PCR and Western blot

  • Verify cellular localization using immunofluorescence

  • Use multiple independent clones when working with stable cell lines

  • Consider the impact of cell density and passage number on expression levels

What advanced techniques can be employed to study AGPAT9 at the molecular level?

For researchers seeking to investigate AGPAT9 at a more sophisticated molecular level, several advanced techniques have proven valuable:

Molecular and structural analysis techniques:

• Protein-protein interaction studies:

  • Co-immunoprecipitation to identify interaction partners

  • Proximity ligation assay (PLA) for in situ detection of protein interactions

  • FRET (Fluorescence Resonance Energy Transfer) for studying dynamic protein interactions

• Functional domain analysis:

  • Creation of truncated proteins to identify functional domains

• Enzymatic activity assessment:

  • In vitro AGPAT enzymatic activity assays using radiolabeled substrates

  • Measurement of lipid metabolite production using mass spectrometry

• Dynamic cellular localization:

  • Live-cell imaging using GFP-tagged AGPAT9

  • FRAP (Fluorescence Recovery After Photobleaching) to study protein mobility

• Transcriptional regulation:

  • Chromatin immunoprecipitation (ChIP) to identify transcription factors regulating AGPAT9

  • Luciferase reporter assays to study promoter activity

What emerging technologies might enhance AGPAT9 antibody applications in cancer research?

Several emerging technologies hold promise for advancing AGPAT9 antibody applications in cancer research:

• Single-cell proteomics:

  • Mass cytometry (CyTOF) incorporating AGPAT9 antibodies for analysis of heterogeneous tumor populations

  • Single-cell Western blotting to assess AGPAT9 expression variability within tumor samples

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM, PALM) for detailed subcellular localization studies

  • Expansion microscopy to visualize AGPAT9 distribution and interactions at nanoscale resolution

• Antibody engineering:

  • Development of recombinant antibody fragments (Fab, scFv) with improved tissue penetration

  • Bispecific antibodies targeting AGPAT9 and other markers for enhanced specificity

  • Leveraging generative AI models for antibody design targeting specific AGPAT9 epitopes

• In situ analysis:

  • Spatial transcriptomics combined with AGPAT9 protein detection for correlation of expression patterns with tissue architecture

  • Multiplexed ion beam imaging (MIBI) for simultaneous detection of multiple proteins including AGPAT9

• Therapeutic applications:

  • Development of antibody-drug conjugates targeting AGPAT9 in cancer cells with aberrant expression

  • PROTAC (Proteolysis Targeting Chimera) approaches for selective AGPAT9 degradation

How might AGPAT9 antibodies contribute to understanding the relationship between lipid metabolism and cancer?

AGPAT9/GPAT3 functions in lipid metabolism pathways, and emerging evidence suggests connections between altered lipid metabolism and cancer progression. AGPAT9 antibodies can facilitate several research approaches:

• Metabolic pathway analysis:

  • Co-localization studies with other enzymes in the glycerophospholipid synthesis pathway

  • Correlation of AGPAT9 expression with lipid profiles in cancer cells

  • Investigation of lipid droplet formation and AGPAT9 localization

• Tumor microenvironment studies:

  • Analysis of AGPAT9 expression in cancer-associated adipocytes

  • Investigation of lipid transfer between adipocytes and cancer cells

  • Correlation between AGPAT9 levels and extracellular pH in tumor microenvironments

• Signaling pathway integration:

  • Exploring how AGPAT9-mediated changes in membrane lipid composition affect oncogenic signaling pathways

  • Investigating potential cross-talk between AGPAT9 and the Wnt/β-catenin pathway

  • Examining relationships between lipid metabolism enzymes and V-ATPase activity

• Therapeutic targeting:

  • Development of small molecule inhibitors of AGPAT9 enzymatic activity

  • Investigation of combination therapies targeting both lipid metabolism and established cancer pathways