LPAR2 Antibody, HRP conjugated

What is LPAR2 and what cellular functions does it mediate?

LPAR2 (lysophosphatidic acid receptor 2) is a G-protein coupled receptor that functions as a receptor for lysophosphatidic acid (LPA), mediating diverse cellular activities. In humans, the canonical protein has 348 amino acid residues with a molecular mass of 38.7 kDa and is primarily localized in the cell membrane . LPAR2 belongs to the G-protein coupled receptor 1 family and is also known by several synonyms including EDG4, LPA-2, and LPA2 . The receptor plays critical roles in cell proliferation, migration, and survival pathways, with recent research highlighting its importance in maintaining endothelial barrier integrity in acute lung injury models .

What is the tissue distribution pattern of LPAR2?

LPAR2 exhibits a distinct expression pattern across human tissues. It is most abundantly expressed in testes and peripheral blood leukocytes, with lower expression levels detected in the pancreas, spleen, thymus, and prostate . This differential expression pattern suggests tissue-specific functions of LPAR2 and should be considered when designing experiments targeting this receptor. When evaluating antibody performance in specific tissue types, researchers should account for these varying expression levels to establish appropriate detection thresholds.

What are the primary applications for LPAR2 Antibody, HRP conjugated?

The HRP-conjugated LPAR2 antibody is specifically optimized for several immunodetection techniques:

The HRP conjugation eliminates the need for secondary antibody incubation, reducing experimental time and potential cross-reactivity issues . For each application, optimization of antibody concentration against your specific sample type is recommended to maximize signal-to-noise ratio.

How should researchers optimize LPAR2 Antibody, HRP conjugated for Western blot applications?

Optimization of Western blot protocols for LPAR2 detection requires careful consideration of several parameters:

• Sample preparation: LPAR2 is a membrane protein, requiring effective membrane protein extraction methods. Lysis buffers containing 1-2% non-ionic detergents (Triton X-100 or NP-40) effectively solubilize LPAR2 while preserving epitope integrity.

• Reducing conditions: As LPAR2 contains multiple cysteine residues that may form disulfide bonds, compare reducing and non-reducing conditions to determine optimal epitope exposure.

• Transfer parameters: Extended transfer times (90-120 minutes) or semi-dry transfer systems may improve transfer efficiency of this transmembrane protein.

• Blocking optimization: Test 5% BSA versus 5% non-fat dry milk in TBS-T to minimize background while preserving specific binding.

• Signal development: For HRP-conjugated antibodies, chemiluminescent substrates with varying sensitivity levels should be tested to optimize detection without signal saturation.

When troubleshooting, compare results against positive control lysates from tissues known to express high LPAR2 levels (testes, leukocytes) and consider using recombinant LPAR2 protein as a standard .

What considerations should researchers take when using LPAR2 Antibody, HRP conjugated in immunohistochemistry?

For optimal immunohistochemical detection of LPAR2 using HRP-conjugated antibodies:

• Fixation evaluation: Compare results between formalin-fixed paraffin-embedded (FFPE) and frozen sections, as membrane protein epitopes can be differentially preserved.

• Antigen retrieval: Test multiple antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0, enzymatic retrieval) to determine optimal epitope exposure.

• Permeabilization: Since LPAR2 is a membrane protein, mild permeabilization (0.1-0.3% Triton X-100) may enhance antibody accessibility without disrupting tissue architecture.

• Signal amplification: For low-abundance expression, consider tyramide signal amplification (TSA) systems compatible with HRP-conjugated antibodies.

• Counterstaining compatibility: Certain counterstains may mask HRP-developed signals; optimize counterstaining intensity accordingly.

Given LPAR2's variable tissue expression, include positive control tissues (testes, leukocyte-rich tissues) and negative controls (antibody diluent only) in each experiment to validate staining specificity .

How can researchers effectively validate the specificity of LPAR2 Antibody, HRP conjugated?

Comprehensive validation of LPAR2 antibody specificity involves multiple complementary approaches:

• Genetic validation: Compare staining patterns between wild-type samples and those from Lpar2 knockout models. Recent studies utilizing Lpar2-/- mice demonstrate the utility of this approach for validation .

• Peptide competition: Pre-incubate antibody with excess immunizing peptide to confirm signal reduction in positive samples.

• Orthogonal detection methods: Correlate protein detection with mRNA expression using RT-PCR or RNA-seq data from the same samples.

• Multiple antibody validation: Compare results with antibodies targeting different LPAR2 epitopes to confirm consistent localization patterns.

• Heterologous expression: Test antibody reactivity in cell lines with controlled LPAR2 overexpression versus non-expressing controls.

How can LPAR2 Antibody, HRP conjugated be employed to study endothelial barrier function in acute lung injury models?

Recent research demonstrates that LPAR2 plays a protective role in maintaining endothelial barrier integrity during septic acute lung injury (ALI). When designing experiments to investigate this pathway:

• Experimental models: Compare lipopolysaccharide (LPS)-induced ALI models in wild-type versus Lpar2-/- mice, assessing parameters such as vascular permeability, inflammatory cell infiltration, and cytokine production .

• Endothelial permeability assays: Implement in vivo permeability assays using Evans blue dye extravasation to quantify barrier function differences, as documented in recent studies showing increased leakage in Lpar2-deficient mice .

• Mechanistic pathway analysis: Employ the HRP-conjugated LPAR2 antibody in combination with other pathway-specific antibodies to investigate the PLC-PKC-FAK signaling cascade through which LPAR2 mediates barrier protection .

• Therapeutic intervention assessment: Evaluate potential LPAR2-targeting therapeutic compounds (such as DBIBB) using the antibody to correlate receptor engagement with functional outcomes in lung injury models .

Data interpretation should account for the complex interplay between LPAR2 activation and downstream effects on tight junction proteins (ZO-1, claudin-5) and adherens junction proteins (VE-cadherin) as demonstrated in recent publications .

What are the considerations for multiplexed detection using LPAR2 Antibody, HRP conjugated alongside other markers?

Multiplexed protein detection involving LPAR2 requires careful experimental design:

• Sequential detection strategies: For HRP-conjugated antibodies in multiplexed IHC, implement sequential detection with complete HRP inactivation between rounds using hydrogen peroxide treatment.

• Chromogen selection: When using multiple HRP-conjugated antibodies, select chromogens with distinct spectral properties (DAB, AEC, TMB) for clear signal discrimination.

• Compatible fixation methods: Identify fixation protocols that preserve epitopes for all target proteins in the multiplexed panel.

• Cross-reactivity testing: Validate the absence of cross-reactivity between antibodies in the multiplex panel through single-staining controls.

• Digital analysis parameters: For quantitative assessment, establish non-overlapping spectral parameters for each chromogen to allow accurate digital separation of signals.

This approach enables simultaneous visualization of LPAR2 with binding partners or downstream signaling components in the same tissue section.

How can researchers optimize detection sensitivity for low-abundance LPAR2 expression?

For tissues with low LPAR2 expression (e.g., pancreas, spleen), sensitivity enhancement strategies include:

• Signal amplification systems: Implement tyramide signal amplification (TSA) compatible with HRP-conjugated antibodies, which can increase detection sensitivity 10-100 fold.

• Extended primary antibody incubation: Overnight incubation at 4°C may enhance binding to low-abundance targets without increasing background.

• Substrate selection: Compare standard DAB with more sensitive alternatives like DAB-Ni or Nova Red for chromogenic applications, or use enhanced chemiluminescent substrates for Western blotting.

• Sample enrichment techniques: Consider membrane protein enrichment protocols or immunoprecipitation prior to Western blot analysis.

• Digital enhancement: Employ computational image analysis with background correction algorithms to reveal subtle expression patterns.

These approaches should be systematically evaluated to determine the optimal combination for specific experimental contexts while maintaining specificity.

What are common issues with LPAR2 Antibody, HRP conjugated and their solutions?

When troubleshooting, implement systematic changes to one variable at a time while maintaining appropriate controls to identify the specific factor affecting performance .

How should researchers interpret conflicting results between LPAR2 detection methods?

When faced with discrepancies between different detection methods:

• Epitope accessibility differences: Different techniques (WB, IHC, IF) expose epitopes differently. Western blotting denatures proteins, potentially revealing epitopes masked in native confirmation used in IF/IHC.

• Isoform specificity: Confirm which LPAR2 isoforms or post-translational modifications your antibody recognizes, as these may vary between tissue types or experimental conditions.

• Technical validation: Verify antibody performance in each application through positive and negative controls, especially when translating between techniques.

• Cross-methodology correlation: When possible, correlate protein detection with mRNA expression data to resolve discrepancies.

• Literature comparison: Review published findings using similar antibodies to identify consensus patterns and potential technical artifacts.

How is LPAR2 research advancing our understanding of septic acute lung injury?

Recent studies using Lpar2 knockout mice have revealed crucial insights into septic acute lung injury pathophysiology:

• Barrier protection mechanism: Lpar2 deficiency significantly increased vascular endothelial permeability in LPS-induced septic models, with knockout mice showing:

  • Increased mortality (58.3% vs. 29.2% in wild-type mice)

  • Enhanced lung edema (higher wet-to-dry ratio)

  • Greater Evans blue dye extravasation

  • Elevated inflammatory cytokine production (IL-6, TNF-α, CXCL15)

• Molecular pathway identification: LPA-LPA2 signaling protects endothelial barrier integrity via the PLC-PKC-FAK pathway, maintaining expression of tight junction proteins (ZO-1, claudin-5) and adherens junction protein VE-cadherin .

• Therapeutic potential: Pharmacological activation of LPAR2 by the agonist DBIBB significantly reduced inflammation, edema, and hemorrhage while increasing expression of vascular endothelial junction proteins in septic models .

These findings suggest that LPAR2-targeted therapies may represent a novel approach for septic ALI treatment, focusing on endothelial barrier protection rather than purely anti-inflammatory mechanisms.

What novel methodological approaches are emerging for LPAR2 protein quantification and localization?

Emerging technologies enhancing LPAR2 research include:

• Super-resolution microscopy: Techniques such as STORM and PALM enable nanoscale visualization of LPAR2 membrane distribution and colocalization with signaling partners at resolutions below the diffraction limit.

• Proximity ligation assays (PLA): These methods can detect protein-protein interactions between LPAR2 and downstream effectors with single-molecule sensitivity in situ.

• Mass spectrometry-based quantification: Targeted proteomics approaches allow absolute quantification of LPAR2 protein levels across different tissues or experimental conditions.

• CRISPR-Cas9 engineered reporter systems: Endogenous tagging of LPAR2 enables live-cell imaging of receptor dynamics without overexpression artifacts.

• Single-cell proteomics: Emerging methods for protein detection at single-cell resolution can reveal LPAR2 expression heterogeneity within tissues.

These methodological advances are expanding our understanding of LPAR2 biology beyond traditional antibody-based approaches, enabling more precise quantification and spatial analysis.