LPAR6 Antibody, Biotin conjugated

What is LPAR6 and why is it significant in biological research?

LPAR6 (Lysophosphatidic Acid Receptor 6) is a protein belonging to the G-protein coupled receptor 1 family. In humans, this protein has a canonical length of 344 amino acid residues with a molecular mass of approximately 39.4 kDa . LPAR6 binds to oleoyl-L-alpha-lysophosphatidic acid (LPA) and is involved in intracellular cAMP signaling .

Biologically, LPAR6 plays critical roles in:

• Maintenance of hair growth and texture

• Cell membrane signaling processes

• Cancer development, particularly hepatocellular carcinoma (HCC)

• Immune system function, including T cell activity regulation

The gene is also known by several synonyms, including HYPT8, LAH3, LPA-6, P2RY5, and P2Y5, which may appear in literature under different research contexts .

What is the structure and composition of LPAR6 Antibody, Biotin conjugated?

LPAR6 Antibody, Biotin conjugated is typically a rabbit polyclonal antibody that has been chemically linked to biotin molecules. Its key characteristics include:

• Host organism: Predominantly rabbit-derived

• Clonality: Polyclonal (recognizes multiple epitopes on the target antigen)

• Conjugation: Biotin, which enables detection through high-affinity binding to streptavidin or avidin systems

• Target region: Often directed against specific amino acid sequences, such as AA 292-313 in the human LPAR6 protein

• Purity: Generally >95%, purified by protein G chromatography

• Storage buffer: Typically preserved in PBS (pH 7.4) with proclin-300 and glycerol

The antibody is designed to recognize specific epitopes on the LPAR6 protein with high specificity and sensitivity for research applications.

What are the primary applications of LPAR6 Antibody, Biotin conjugated in research?

The biotin-conjugated LPAR6 antibody has several validated research applications:

The biotin conjugation specifically enhances sensitivity through amplification systems using avidin/streptavidin-HRP complexes, allowing for efficient detection of low-abundance LPAR6 protein in experimental samples . This configuration is particularly advantageous for tissue staining applications where signal amplification is beneficial.

How can LPAR6 Antibody, Biotin conjugated be used to characterize LPA signaling pathways in cancer research?

LPAR6 plays a significant role in cancer progression, particularly in hepatocellular carcinoma (HCC). Research methodologies employing biotin-conjugated LPAR6 antibodies can effectively:

• Quantify LPAR6 overexpression: Studies have demonstrated that LPAR6 overexpression in HCC specimens correlates with poor patient survival . Using biotin-conjugated antibodies in immunohistochemistry applications allows researchers to quantitatively assess LPAR6 expression levels in tumor samples compared to normal tissues.

• Investigate downstream signaling components: Research has identified that LPAR6 activates STAT3-dependent mechanisms leading to upregulation of Pim-3, a relevant target gene in HCC tumorigenesis . Biotin-conjugated antibodies can be employed in co-immunoprecipitation studies followed by protein complex detection to map these interactions.

• Evaluate experimental therapeutic interventions: When testing LPA signaling inhibitors, biotin-conjugated LPAR6 antibodies can monitor receptor downregulation or internalization using immunofluorescence approaches.

• Correlate LPAR6 expression with clinical parameters: Using biotin-conjugated antibodies in tissue microarray analyses allows high-throughput screening of LPAR6 expression across multiple patient samples to establish correlations with tumor grade, proliferation rates, and survival outcomes .

Methodologically, these applications often employ a streptavidin-biotin amplification system to enhance detection sensitivity, making biotin-conjugated LPAR6 antibodies particularly valuable for detecting subtle changes in receptor expression or localization.

What are the challenges in optimizing LPAR6 Antibody, Biotin conjugated for dual immunofluorescence staining?

Dual immunofluorescence involving biotin-conjugated LPAR6 antibody presents several technical challenges that researchers must address:

• Endogenous biotin interference: Tissues often contain endogenous biotin which can cause high background signals. Methodological solution: Include a biotin blocking step using avidin followed by biotin blocking reagents prior to antibody application.

• Cross-reactivity with other primary antibodies: When conducting dual labeling, potential cross-reactivity between antibodies must be considered. Methodological approach: Sequential rather than simultaneous incubation may be necessary, with the LPAR6 biotin-conjugated antibody typically applied first, followed by thorough washing before the second primary antibody.

• Signal amplification balance: Biotin-streptavidin systems provide significant amplification which may overwhelm other fluorescence signals. Optimization strategy: Titration experiments should be conducted to determine the optimal concentration of biotin-conjugated LPAR6 antibody (typically starting at 1:50-1:200 dilutions) .

• Spectral overlap: When selecting fluorophores for detection of the biotin-streptavidin complex, consider potential spectral overlap with other fluorescent markers. Solution: Choose streptavidin conjugates with fluorophores spectrally distant from other markers in the experiment.

• Tissue autofluorescence: Particularly in liver and brain tissues where LPAR6 research is common, autofluorescence can interfere with specific signals. Mitigation approach: Include Sudan Black B (0.1-0.3%) treatment post-fixation to reduce lipofuscin-related autofluorescence.

A systematic optimization protocol should include antibody dilution series (1:50, 1:100, 1:200) , varied incubation times (1-18 hours), and comparison of different streptavidin-fluorophore conjugates to achieve optimal signal-to-noise ratio.

How can proteome-wide profiling techniques be integrated with LPAR6 Antibody, Biotin conjugated for identification of novel interaction partners?

Integration of biotin-conjugated LPAR6 antibodies with advanced proteomics approaches offers powerful strategies for discovering novel interaction partners:

• Co-immunoprecipitation coupled with mass spectrometry: Biotin-conjugated LPAR6 antibodies can be used to pull down LPAR6 along with its interaction partners from cell lysates. The biotin tag allows efficient capture on streptavidin beads, followed by mass spectrometry analysis to identify the co-precipitated proteins. This approach can reveal transient or weak interactions not detectable by traditional methods .

• Proximity-dependent biotin identification (BioID): By fusing a promiscuous biotin ligase (BirA*) to LPAR6, proteins in close proximity become biotinylated. These can then be captured using streptavidin and identified by mass spectrometry. The biotin-conjugated LPAR6 antibody serves as a control to validate the expression and localization of the fusion protein .

• Crosslinking mass spectrometry (XL-MS): Chemical crosslinking of LPAR6 with its interaction partners, followed by enrichment using biotin-conjugated LPAR6 antibodies and mass spectrometry analysis, can provide structural insights into protein complexes involving LPAR6.

• SILAC-based quantitative proteomics: As demonstrated in research on LPA-binding proteins, SILAC (stable isotope labeling by amino acids in cell culture) combined with biotin-conjugated antibody enrichment allows quantitative assessment of binding affinities between LPAR6 and potential interaction partners .

An example protocol employing this integration includes:

• Cell lysis in mild detergent buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.5)

• Pre-clearing lysates with protein G beads

• Incubation with biotin-conjugated LPAR6 antibody (5-10 μg per mg of lysate)

• Capture with streptavidin-coated magnetic beads

• Rigorous washing (including high salt and detergent washes)

• On-bead tryptic digestion

• LC-MS/MS analysis

• Data filtering using appropriate statistical approaches to identify high-confidence interaction partners

This integrated approach has successfully identified 86 candidate LPA-binding proteins in HEK293T cells , demonstrating the power of combining antibody-based enrichment with proteomics technologies.

What is the optimal protocol for using LPAR6 Antibody, Biotin conjugated in immunohistochemistry of formalin-fixed paraffin-embedded tissues?

The following protocol represents an optimized methodology for using biotin-conjugated LPAR6 antibody in FFPE tissues:

Materials required:

• Biotin-conjugated LPAR6 antibody

• Streptavidin-HRP conjugate

• DAB substrate kit

• Antigen retrieval buffer (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Hydrogen peroxide (3%)

• Normal serum (from same species as secondary antibody)

• PBS with 0.1% Tween-20 (PBST)

• Hematoxylin (for counterstaining)

Protocol:

• Deparaffinization and rehydration:

  • Incubate slides at 60°C for 1 hour

  • Process through xylene (3 × 5 minutes) and graded alcohols (100%, 95%, 70%)

  • Rinse in distilled water

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) at 95-98°C for 20 minutes

  • Allow to cool to room temperature (approximately 20 minutes)

  • Rinse in PBST (3 × 5 minutes)

• Endogenous peroxidase and biotin blocking:

  • Block endogenous peroxidase with 3% H₂O₂ in methanol for 10 minutes

  • Rinse in PBST (3 × 5 minutes)

  • Apply avidin solution for 15 minutes

  • Rinse briefly with PBST

  • Apply biotin solution for 15 minutes

  • Rinse in PBST (3 × 5 minutes)

• Protein blocking:

  • Block with 5% normal serum in PBST for 1 hour at room temperature

  • Drain blocking solution (do not rinse)

• Primary antibody incubation:

  • Apply biotin-conjugated LPAR6 antibody at 1:200-1:500 dilution

  • Incubate at 4°C overnight (12-16 hours)

  • Rinse in PBST (3 × 5 minutes)

• Streptavidin-HRP incubation:

  • Apply streptavidin-HRP conjugate (1:100-1:500 dilution)

  • Incubate for 30 minutes at room temperature

  • Rinse in PBST (3 × 5 minutes)

• Visualization:

  • Apply DAB substrate solution for 5-10 minutes (monitor microscopically)

  • Rinse in distilled water

• Counterstaining and mounting:

  • Counterstain with hematoxylin for 30 seconds

  • Rinse in running tap water

  • Dehydrate through graded alcohols and clear in xylene

  • Mount with permanent mounting medium

Critical optimization parameters:

• Antibody dilution should be determined empirically for each lot and tissue type

• Antigen retrieval conditions may need adjustment based on fixation time

• Incubation time can be extended to 48 hours for detection of low abundance targets

• For dual staining applications, complete this protocol first, then apply a different detection system for the second primary antibody

As noted in the literature, this method has been effectively used in studies validating the connection between LPAR6 overexpression and PIM-3 in hepatocellular carcinoma specimens .

How can researchers optimize Western blot protocols for LPAR6 Antibody, Biotin conjugated to detect low abundance receptor expression?

Detecting low abundance membrane proteins like LPAR6 requires specific optimizations when using biotin-conjugated antibodies in Western blot applications:

Optimized Protocol:

• Sample preparation:

  • Extract total membrane proteins using a specialized membrane protein extraction kit

  • Add protease inhibitor cocktail immediately after cell lysis

  • For LPAR6 enrichment, consider using ConA-sepharose to pre-enrich glycosylated membrane proteins

• Protein quantification and loading:

  • Load 50-80 μg of total protein per lane (higher than standard 20-30 μg)

  • Include positive control lysates from cells with confirmed LPAR6 overexpression

• Gel electrophoresis:

  • Use 10-12% polyacrylamide gels for optimal resolution of the 39.4 kDa LPAR6 protein

  • Run at lower voltage (80-100V) to improve protein resolution

• Transfer optimization:

  • Use PVDF membrane (0.2 μm pore size) instead of nitrocellulose

  • Transfer at 30V overnight at 4°C for better transfer of hydrophobic membrane proteins

  • Verify transfer efficiency with reversible protein stain

• Blocking:

  • Block with 5% BSA in TBST (not milk, which contains biotin)

  • Include 0.05% SDS in blocking buffer to enhance accessibility of epitopes

• Primary antibody incubation:

  • Dilute biotin-conjugated LPAR6 antibody at 1:1000-1:2000

  • Incubate overnight at 4°C with gentle rocking

  • Consider adding 0.01% SDS to antibody dilution buffer

• Detection system:

  • Use high-sensitivity streptavidin-HRP conjugate (1:2000-1:5000)

  • Incubate for 1 hour at room temperature

  • Wash extensively (5 × 5 minutes with TBST)

• Signal development:

  • Use enhanced chemiluminescence substrate formulated for low abundance proteins

  • Extend exposure times incrementally (30 seconds to 10 minutes)

  • Consider using a digital imager with cooling capabilities for long exposures

Troubleshooting low signal issues:

• If no signal is detected, perform dot blot analysis to confirm antibody binding activity

• Consider including a deglycosylation step if glycosylation shields epitope recognition

• Test membrane stripping and reprobing with a different LPAR6 antibody targeting a different epitope to confirm results

This optimized protocol has been validated for detecting LPAR6 in various mammalian cell lines and tissues including human, mouse, rat, and other species with high sequence homology to the target epitope .

What controls should be included when using LPAR6 Antibody, Biotin conjugated in flow cytometry experiments?

Flow cytometry experiments using biotin-conjugated LPAR6 antibody require comprehensive controls to ensure reliable and interpretable data:

Essential Controls:

• Unstained cells control:

  • Purpose: Establishes baseline autofluorescence of the cell population

  • Implementation: Process cells through all steps except antibody incubation

• Isotype control:

  • Purpose: Detects non-specific binding of the antibody's constant region

  • Implementation: Use biotin-conjugated rabbit IgG at the same concentration as the LPAR6 antibody

  • Interpretation: Signal from test samples should be significantly higher than isotype control

• Streptavidin-only control:

  • Purpose: Identifies potential direct binding of streptavidin-fluorophore to cells

  • Implementation: Omit primary antibody but include streptavidin-fluorophore step

• Cells with known LPAR6 expression levels:

  • Positive control: Cell line with confirmed high LPAR6 expression (e.g., certain HCC cell lines )

  • Negative control: Cell line with confirmed absence of LPAR6 expression

  • Implementation: Process alongside test samples

• FMO (Fluorescence Minus One) controls:

  • Purpose: Essential for multicolor panels to set proper gating boundaries

  • Implementation: Include all fluorochromes except the one conjugated to streptavidin

• Biological controls:

  • LPAR6 knockdown/knockout cells: Demonstrates antibody specificity

  • LPA-stimulated cells: Shows dynamic range of detection if receptor internalization occurs

• Technical controls:

  • Titration series: Test multiple dilutions of the biotin-conjugated LPAR6 antibody to determine optimal signal-to-noise ratio

  • Blocking control: Pre-incubate antibody with immunizing peptide to confirm specificity

Data Analysis Considerations:

• Report median fluorescence intensity (MFI) rather than percent positive, particularly for receptors like LPAR6 that may have variable expression levels

• Calculate signal-to-noise ratio by dividing the MFI of the sample by the MFI of the isotype control

• For quantitative applications, include calibration beads to convert arbitrary fluorescence units to antibody binding capacity (ABC)

This comprehensive control strategy has been validated in studies examining T cell function modulation by LPA signaling, where precise quantification of receptor expression was critical for interpreting functional outcomes .

How has LPAR6 Antibody, Biotin conjugated contributed to understanding the role of LPAR6 in hepatocellular carcinoma (HCC)?

Biotin-conjugated LPAR6 antibodies have played a crucial role in elucidating the mechanisms through which LPAR6 contributes to hepatocellular carcinoma development:

• Expression profiling and prognostic significance:

  • Research using biotin-conjugated LPAR6 antibodies in immunohistochemical analyses has demonstrated that LPAR6 is significantly overexpressed in HCC specimens compared to adjacent non-tumorous tissues

  • Studies of 128 HCC patients revealed that high LPAR6 expression correlates with poor survival outcomes

  • The biotin-conjugation allowed for sensitive detection and quantification of expression levels across tissue microarrays

• Molecular mechanism identification:

  • LPAR6 was found to upregulate Pim-3 (a serine/threonine kinase) through a STAT3-dependent mechanism

  • Using biotin-conjugated antibodies in chromatin immunoprecipitation experiments helped establish this transcriptional regulatory relationship

  • The pathway was further validated through co-expression analysis in clinical specimens

• Functional validation in tumor models:

  • Studies employing biotin-conjugated LPAR6 antibodies for detection in xenograft experiments demonstrated that RNAi-mediated attenuation of LPAR6 impaired HCC tumorigenicity in vivo

  • Immunohistochemical analysis of xenograft tissues using biotin-conjugated antibodies showed reduced proliferation markers in LPAR6-depleted tumors

• Therapeutic target evaluation:

  • The characterization of LPAR6 as a tumor-promoting factor using biotin-conjugated antibody detection methods has positioned it as a potential therapeutic target

  • Monitoring LPAR6 expression levels in response to experimental therapies has provided insights into the molecular mechanisms of drug efficacy

These findings collectively establish LPAR6 as both a prognostic biomarker and a potential therapeutic target in HCC, with biotin-conjugated antibodies serving as essential tools in this research .

What role does LPAR6 play in T cell immunobiology and how have antibody-based detection methods advanced this understanding?

LPAR6 has emerged as a significant modulator of T cell function, with antibody-based detection methods revealing critical insights into immune regulation:

• Impact on immune synapse formation:
  Research using immunofluorescence techniques with LPAR6 antibodies has demonstrated that LPA signaling through LPAR6 disrupts the formation of the immune synapse between CD8+ T cells and their target cells . Specifically:

  • LPA impairs the localization of IP3R1 (inositol 1,4,5-trisphosphate receptor type 1) to the immune synapse

  • Cytoskeletal rearrangements necessary for effective T cell function are disrupted

  • Both actin polymerization and microtubule detyrosination are negatively affected

• Mechanism of immune suppression:
  Antibody-based intracellular staining protocols have revealed that LPA alters RhoA activity beyond optimal TCR-induced levels :

  • LPA causes an increase in early RhoA activity that exceeds TCR-induced RhoA activation

  • This disrupts the precise balance of RhoA activity required for mDia1 and IFNγ localization

  • The end result is reduced calcium store release and impaired directed cytokine release

• Impact on cytokine production and release:
  Using flow cytometry with intracellular cytokine staining, researchers demonstrated that:

  • LPA signaling through LPAR6 impairs TCR-induced IL-2 and IFNγ secretion

  • The mechanism involves altered calcium signaling downstream of TCR activation

  • These effects contribute to immune suppression in the tumor microenvironment

• Therapeutic implications:
  The characterization of LPAR6's role in T cell function suggests potential immunotherapeutic applications:

  • Targeting LPAR6 may enhance anti-tumor T cell responses

  • Modulating LPA levels in the tumor microenvironment could potentially overcome immunosuppression

  • Monitoring LPAR6 expression on tumor-infiltrating lymphocytes may provide prognostic information

These findings highlight the importance of LPAR6 in regulating immune responses and suggest that targeting this pathway may have implications for cancer immunotherapy strategies .

How can chemical proteomic profiling be combined with LPAR6 antibody detection to identify novel LPA-binding proteins?

The integration of chemical proteomic profiling with LPAR6 antibody detection represents a powerful approach for discovering novel LPA-binding proteins:

• LPA probe-based proteome labeling strategy:
  Researchers have developed desthiobiotin-conjugated LPA acyl phosphate probes that covalently label LPA-binding proteins . This approach:

  • Utilizes an LPA-affinity probe with three components: LPA (targeting the binding pocket), desthiobiotin (for enrichment), and a linker

  • Enables proteome-wide identification of proteins that interact with LPA

  • Has successfully identified 939 putative LPA-binding proteins from two different cell lines

• SILAC-based quantitative workflow:
  By combining stable isotope labeling with LPA probe concentrations, researchers can differentiate specific from non-specific interactions :

  • Low (10 μM) and high (100 μM) concentrations of LPA probe react with light- and heavy-labeled cell lysates

  • Relative binding affinities (RLPA10/1) are calculated to identify proteins with specific LPA-binding capabilities

  • This approach identified 86 proteins with highly selective interactions with LPA

• Validation using LPAR6 antibodies:
  After identifying candidate LPA-binding proteins through chemical proteomics, biotin-conjugated LPAR6 antibodies can be employed to:

  • Confirm co-localization of identified proteins with LPAR6 through dual immunofluorescence

  • Validate protein-protein interactions through co-immunoprecipitation experiments

  • Assess competitive binding between LPA and the newly identified proteins using proximity ligation assays

• Application to other phospholipid-binding proteins:
  The researchers note that this methodology can be adapted for studying other lipid-binding proteins at the proteome-wide scale :

  • Similar acyl phosphate probes can be designed for phospholipids carrying terminal phosphate groups

  • Integration with antibody-based detection methods allows for comprehensive validation

  • This approach overcomes limitations of traditional methods in identifying transient or weak interactions

This integrated approach represents a significant advancement in our ability to map the complete interactome of LPA signaling networks, with important implications for understanding LPAR6 function in various biological contexts .

What are the key challenges and solutions in using LPAR6 Antibody, Biotin conjugated for studying membrane protein complexes?

Investigating LPAR6-containing membrane protein complexes using biotin-conjugated antibodies presents several technical challenges that researchers have developed solutions to address:

1. Membrane protein solubilization challenges:

2. Epitope accessibility issues:

3. Detection sensitivity limitations:

4. Validation of complex components:

These methodological refinements have enabled researchers to characterize LPAR6-containing protein complexes in various cellular contexts, significantly advancing our understanding of LPA signaling networks and identifying potential therapeutic targets in conditions where these pathways are dysregulated.