LPCAT3 Antibody

What is LPCAT3 and why are antibodies against it important for research?

LPCAT3 (Lysophosphatidylcholine acyltransferase 3) is a crucial enzyme in phospholipid remodeling, also known as the Lands cycle. This 56 kDa protein catalyzes the reacylation step in phospholipid metabolism by transferring fatty acyl chains from fatty acyl-CoA to lysophospholipids, forming various phospholipid classes . LPCAT3 is particularly important as it preferentially incorporates arachidonate into phosphatidylcholine (PC) in endoplasmic reticulum membranes, which influences membrane dynamics and enables triacylglycerol transfer to nascent lipoproteins . Antibodies against LPCAT3 are essential research tools for studying lipid metabolism, membrane composition regulation, and related pathological conditions.

What alternative names should researchers be aware of when searching for LPCAT3 antibodies?

When searching literature or antibody databases, researchers should be aware that LPCAT3 may be referenced under several alternative designations including:

• C3F

• MBOAT5 (Membrane-bound O-acyltransferase domain-containing protein 5)

• LPCAT (when referring specifically to this isoform)

• LPLAT 5 (Lysophospholipid acyltransferase 5)

• LPSAT (Lysophosphatidylserine acyltransferase)

These alternative names reflect the evolving understanding of this protein's function and its relationship to other acyltransferases .

What are the typical applications of LPCAT3 antibodies in research?

LPCAT3 antibodies are versatile tools employed in multiple experimental applications:

• Western Blot (WB): The most common application for detecting LPCAT3 protein expression levels and molecular weight

• Immunohistochemistry (IHC): For examining tissue distribution and localization patterns

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of LPCAT3

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

• Immunoprecipitation (IP): For protein-protein interaction studies or purification prior to other analyses

The application versatility allows researchers to investigate LPCAT3 from multiple experimental angles .

What species reactivity do LPCAT3 antibodies typically exhibit?

Available LPCAT3 antibodies demonstrate reactivity against various species:

• Human LPCAT3: Most commonly available and extensively characterized

• Mouse LPCAT3: Essential for murine model studies of lipid metabolism

• Rat LPCAT3: Used in rat model systems

• Porcine LPCAT3: Some antibodies show cross-reactivity with pig samples

When selecting an antibody, researchers should verify the specific species reactivity needed for their experimental system, as not all antibodies work across all species despite sequence homology .

What forms of LPCAT3 antibodies are available and how should researchers choose between them?

Researchers can select from several LPCAT3 antibody formats based on experimental needs:

• Host Species:

  • Rabbit polyclonal: Offers broad epitope recognition but potential batch variability

  • Mouse monoclonal: Provides consistent specificity for particular epitopes

• Conjugation Options:

  • Unconjugated: Versatile for most applications requiring secondary antibody detection

  • HRP-conjugated: Direct detection in ELISA and Western blot without secondary antibodies

  • FITC-conjugated: Direct fluorescent detection in microscopy applications

  • Biotin-conjugated: Enhanced signal amplification through avidin/streptavidin systems

Selection should be based on the specific experimental application, detection system availability, and need for multiplexing with other antibodies .

How should researchers validate LPCAT3 antibody specificity?

Rigorous validation of LPCAT3 antibody specificity is critical and should include:

• Positive and negative controls:

  • LPCAT3 overexpression systems (such as FLAG-tagged murine LPCAT3 in RH 7777 cells)

  • LPCAT3-knockout cells generated using CRISPR/Cas9 technology

  • Rescue experiments with wild-type and enzymatically inactive mutant (H374A) LPCAT3

• Cross-validation methods:

  • Use of multiple antibodies targeting different epitopes of LPCAT3

  • Correlation of protein detection with mRNA expression data

  • Tag-based detection (e.g., Myc-tagged LPCAT3) to confirm specificity of observed bands

• Blocking peptide experiments:

  • Pre-incubation of antibody with immunogen peptide should abolish specific signal

This comprehensive validation approach ensures experimental observations are attributable to LPCAT3 and not off-target effects .

What is the recommended Western blot protocol for LPCAT3 detection?

For optimal LPCAT3 detection by Western blot, researchers should follow this protocol:

• Sample preparation:

  • Harvest cells and prepare lysates in RIPA buffer with protease inhibitors

  • For membrane-enriched fractions: centrifuge cleared lysates at 100,000×g for 1 hour

  • Resuspend membrane pellet in TSE buffer (20 mM Tris-HCl [pH7.4], 300 mM sucrose, 1 mM EDTA)

  • Determine protein concentration using Bradford assay

• SDS-PAGE and transfer:

  • Resolve 20μg protein on 10% SDS-polyacrylamide gels

  • Transfer to nitrocellulose membrane using semi-dry transfer system

• Antibody incubation:

  • Block membrane overnight with 5% skim milk in TBST

  • Incubate with primary LPCAT3 antibody (40 ng/ml for anti-LPCAT3)

  • Wash thoroughly with TBST (at least three changes)

  • Incubate with HRP-conjugated secondary antibody (1:2000 dilution)

  • Develop using ECL select detection system

• Expected results:

  • LPCAT3 should appear at approximately 56 kDa

  • Potential cleaved forms may appear at lower molecular weights, especially in viral infection studies

This protocol has been validated in multiple studies and provides consistent LPCAT3 detection .

How can researchers establish and validate LPCAT3-deficient cell models?

To establish effective LPCAT3-deficient models:

• CRISPR/Cas9-mediated knockout:

  • Design guide RNAs targeting LPCAT3 coding regions

  • Transfect cells with Cas9 and guide RNA constructs

  • Screen clones by genomic PCR and sequencing to confirm mutations

  • Validate knockout by Western blot and enzymatic activity assays

• Lentiviral shRNA-mediated knockdown:

  • Design shRNA targeting specific LPCAT3 sequences (e.g., GGCTTAAGGTGTACAGATC)

  • Construct lentiviral vectors with puromycin resistance

  • Co-transfect with packaging vectors (VSVG and PXPAX2) in 293T cells

  • Collect, concentrate and titer viral particles

  • Infect target cells and select with puromycin (0.5 μg/ml)

  • Validate knockdown efficiency by qRT-PCR (>80% reduction ideal) and Western blot

• Functional validation:

  • Measure LPCAT enzymatic activity in membrane fractions

  • Analyze phospholipid composition changes by LC-MS

  • Confirm specificity by rescue experiments with wild-type LPCAT3

These approaches provide complementary methods to study LPCAT3 function through loss-of-function models .

How should researchers measure LPCAT3 enzymatic activity?

For accurate assessment of LPCAT3 enzymatic activity:

• Membrane preparation:

  • Isolate membrane fractions by ultracentrifugation (100,000×g)

  • Resuspend in buffer containing 20 mM Tris-HCl, 300 mM sucrose, and 1 mM EDTA

• Activity assay components:

  • Lysophosphatidylcholine (LPC) substrate

  • Various acyl-CoA donors (linoleoyl-CoA and arachidonoyl-CoA)

  • Radiolabeled or fluorescently labeled substrates for quantification

• Analysis methods:

  • Measure incorporation of labeled acyl groups into phospholipids

  • Quantify enzymatic activity as nmol/min/mg protein

  • Compare activity with different acyl-CoA donors to assess substrate selectivity

• Controls and validation:

  • Include wild-type control samples in parallel

  • Use LPCAT3-overexpressing samples as positive controls

  • Employ LPCAT3-knockout samples as negative controls

  • Test selective inhibitors to confirm specificity

This comprehensive approach allows accurate assessment of LPCAT3's contribution to cellular lysophosphatidylcholine acyltransferase activity .

How does LPCAT3 deficiency affect cellular phospholipid composition and what methods are used to analyze these changes?

LPCAT3 deficiency significantly alters cellular phospholipid profiles, particularly affecting arachidonate-containing species:

• Expected phospholipid changes:

  • Decreased levels of arachidonate-containing PC species (36:4 PC and 38:4 PC)

  • Minimal effect on linoleate-containing PC species (34:2 PC)

  • Altered membrane fluidity and physical properties

• Analytical methods:

  • Liquid chromatography-tandem mass spectrometry (LC-MS): Primary method for phospholipid profiling

    • Selected reaction monitoring (SRM) to resolve acyl-chain compositions

    • Chromatographic separation to distinguish isomers (e.g., 38:4 PC isomers)

    • Quantification of specific PC species like 16:0-20:4 PC and 18:0-20:4 PC

• Data interpretation:

  • Compare peaks between wild-type and LPCAT3-deficient samples

  • Examine changes in specific acyl chain combinations

  • Correlate phospholipid changes with functional outcomes

This comprehensive lipidomic analysis reveals LPCAT3's selective role in enriching membranes with arachidonate-containing phospholipids .

What is the relationship between LPCAT3 and viral infections, particularly coronaviruses?

Recent research has revealed important connections between LPCAT3 and viral pathogenesis:

• LPCAT3 cleavage in viral infections:

  • SARS-CoV-2 main protease (Mpro) induces LPCAT3 cleavage

  • Cleavage can be detected as unexpected LPCAT3 fragments by Western blot

  • Mpro-induced cleavage appears dose-dependent on viral protease expression

• Experimental approaches to study this phenomenon:

  • Tag-based detection systems (e.g., Myc-tagged LPCAT3) to confirm specific cleavage

  • Time-course experiments to monitor cleavage progression

  • Protease inhibitor studies to confirm mechanism

  • Direct comparison between infected and uninfected cells

• Functional consequences:

  • LPCAT3 cleavage may contribute to ER stress during infection

  • May relate to gastrointestinal symptoms in coronavirus diseases

  • Potential disruption of membrane phospholipid composition

This research area represents an emerging frontier connecting lipid metabolism to viral pathogenesis mechanisms .

How do researchers differentiate between direct effects of LPCAT3 on membrane composition versus indirect effects on cell signaling pathways?

Distinguishing direct and indirect LPCAT3 effects requires sophisticated experimental approaches:

• Direct membrane composition effects:

  • Comprehensive lipidomic analysis of membrane phospholipids by LC-MS

  • Membrane fluidity measurements using fluorescence anisotropy

  • Reconstitution experiments with purified LPCAT3 in artificial membrane systems

• Cell signaling pathway analysis:

  • Phosphoproteomic analysis to identify altered signaling pathways

  • Investigation of ER stress markers (e.g., PERK, IRE1α, ATF6)

  • Analysis of SREBF1 processing and translocation to Golgi

  • Assessment of inflammatory cytokine production

• Mechanistic separation approaches:

  • Use of catalytically inactive LPCAT3 mutants (H374A) to separate enzymatic from scaffolding functions

  • Temporal analysis distinguishing immediate membrane changes from downstream signaling effects

  • Domain-specific mutations to identify regions involved in protein-protein interactions versus enzymatic activity

These approaches help researchers differentiate LPCAT3's primary role in phospholipid remodeling from its secondary effects on cellular signaling pathways .

What experimental controls are essential when studying LPCAT3's role in triacylglycerol metabolism and lipoprotein assembly?

When investigating LPCAT3's functions in lipid transport:

• Essential controls for lipoprotein assembly studies:

  • Expression analysis of other key proteins: MTP (microsomal triglyceride transfer protein) and PDI (protein disulfide isomerase)

  • Assessment of ER stress markers to rule out indirect effects

  • Measurement of cellular neutral lipid content by enzymatic assays or lipid staining

• Triacylglycerol metabolism analysis approaches:

  • Radiolabeled fatty acid incorporation studies

  • Analysis of triacylglycerol synthesis, storage, and secretion

  • Lipoprotein particle analysis by gradient ultracentrifugation

  • Measurement of apolipoprotein B secretion

• Rescue experiments:

  • Complementation with wild-type LPCAT3

  • Complementation with catalytically inactive LPCAT3

  • Supplementation with specific phospholipid species to bypass LPCAT3 deficiency

This comprehensive control strategy ensures observed phenotypes are specifically attributable to LPCAT3's role in triacylglycerol metabolism and lipoprotein assembly .

What are common issues with LPCAT3 detection by Western blot and how can they be resolved?

Researchers frequently encounter these challenges when detecting LPCAT3:

• Multiple bands or unexpected molecular weights:

  • Potential causes: Post-translational modifications, proteolytic cleavage, viral protease activity

  • Solutions:

    • Include protease inhibitors in all buffers

    • Use freshly prepared samples

    • Compare with tagged LPCAT3 constructs

    • Verify with knockout controls

• Weak or absent signal:

  • Potential causes: Low expression levels, inefficient extraction, antibody sensitivity

  • Solutions:

    • Enrich membrane fractions (100,000×g ultracentrifugation)

    • Optimize blocking conditions (5% milk in TBST recommended)

    • Extend primary antibody incubation (overnight at 4°C)

    • Use enhanced chemiluminescence detection systems

• High background:

  • Potential causes: Insufficient blocking, excessive antibody concentration

  • Solutions:

    • Extend blocking time (overnight recommended)

    • Increase washing steps (minimum three TBST changes)

    • Titrate primary antibody concentration

    • Use monoclonal antibodies for greater specificity

These troubleshooting approaches address the most common technical challenges in LPCAT3 detection .

How can researchers distinguish between specific LPCAT3 cleavage products and non-specific antibody binding?

Differentiating specific cleavage products from non-specific binding requires systematic validation:

• Tag-based verification approach:

  • Express epitope-tagged LPCAT3 (N-terminal Myc-tag recommended)

  • Compare detection with anti-LPCAT3 and anti-tag antibodies

  • Convergent detection of the same fragments confirms specificity

  • Different patterns suggest non-specific binding

• Treatment-dependent confirmation:

  • Monitor fragment appearance in response to specific stimuli (e.g., viral infection)

  • Examine dose-dependency of fragment generation

  • Use appropriate inhibitors to prevent cleavage

  • Correlate fragment appearance with functional consequences

• Mass spectrometry validation:

  • Immunoprecipitate LPCAT3 and analyze fragments by mass spectrometry

  • Identify exact cleavage sites

  • Generate site-directed mutants resistant to cleavage

  • Compare detection patterns with predicted fragment sizes

This systematic approach distinguishes genuine cleavage products from antibody artifacts, particularly important in viral infection studies .

What are emerging applications of LPCAT3 antibodies in disease research?

LPCAT3 antibodies are increasingly valuable for investigating several disease mechanisms:

• Metabolic disorders:

  • Non-alcoholic fatty liver disease (NAFLD)

  • Insulin resistance and diabetes

  • Atherosclerosis and cardiovascular diseases

• Viral infections:

  • Coronavirus pathogenesis mechanisms

  • ER stress responses during infection

  • Viral-induced alterations in lipid metabolism

  • Potential therapeutic targets in the LPCAT3 pathway

• Inflammatory conditions:

  • LPCAT3's role in regulating arachidonic acid availability

  • Connections to eicosanoid production

  • Anti-inflammatory mechanisms of liver X receptor signaling

These emerging applications highlight LPCAT3's expanding significance beyond basic lipid metabolism research .

What technological advances might enhance LPCAT3 antibody applications in the future?

Several technological developments promise to expand LPCAT3 research capabilities:

• Advanced imaging applications:

  • Super-resolution microscopy for precise subcellular localization

  • Live-cell imaging with fluorescently tagged antibody fragments

  • Correlative light and electron microscopy for ultrastructural studies

• Multiplexed detection systems:

  • Mass cytometry (CyTOF) for single-cell protein profiling

  • Imaging mass cytometry for spatial tissue analysis

  • Multiplexed immunofluorescence with spectral unmixing

• Integrated multi-omics approaches:

  • Combined antibody-based proteomics with lipidomics

  • Spatial transcriptomics correlated with protein localization

  • Systems biology modeling of LPCAT3-regulated lipid networks