LOX1.7 Antibody

What are the primary validated applications for LOX-1/OLR1 antibodies in research?

LOX-1/OLR1 antibodies have been extensively validated for multiple applications with specific dilution recommendations. For polyclonal antibodies like 11837-1-AP, validated applications include:

These antibodies have been successfully employed in knockdown/knockout validation, co-immunoprecipitation (CoIP), and immunofluorescence (IF) studies . When using these antibodies, it's essential to optimize dilutions for your specific experimental system and sample type, as reactivity can vary between human and mouse samples.

What is the molecular weight profile of LOX-1/OLR1 in Western blot applications?

When designing Western blot experiments using LOX-1/OLR1 antibodies, researchers should note the discrepancy between calculated and observed molecular weights:

• Calculated molecular weight: 31 kDa (273 amino acids)

• Observed molecular weight range: 40-55 kDa

This discrepancy likely results from post-translational modifications. In prostate cancer cell lines, LOX-1 appears consistently at approximately 40 kDa . The difference between calculated and observed weights is important for proper identification and validation of bands in your Western blot experiments.

What tissue samples and cell lines have been validated for LOX-1/OLR1 antibody detection?

When selecting appropriate positive controls for your experiments, consider these validated samples:

These validated samples provide reliable positive controls for establishing your experimental protocols.

How can LOX-1/OLR1 antibodies be utilized to study immune cell functions?

LOX-1 expression exhibits a cell-specific pattern within the immune system that researchers should consider when designing experiments:

• Expressed on CD1c+ skin dermal DCs and blood myeloid DCs

• Not expressed on Langerhans cells or plasmacytoid DCs

• Expressed on fractions of peripheral B cells and monocytes

When studying immune responses, it's important to note that targeting dendritic cells via LOX-1 can efficiently elicit antigen-specific IFNγ-producing CD4+ T cell responses, promoting Th1-type responses . This makes LOX-1 targeting particularly valuable for research focused on enhancing immunity against cancers and viral infections.

For B cell interaction studies, research has shown that LOX-1+CD11c+ DCs interact with IgD+ B cells in marginal zones of human spleens . Activation of LOX-1 on dendritic cells using agonistic antibodies induces production of BAFF (B cell activating factor) and APRIL (a proliferation inducing ligand), which promote humoral responses by inducing class switching and plasmablast generation .

What are the optimal antigen retrieval protocols for LOX-1/OLR1 immunohistochemistry?

For successful LOX-1/OLR1 immunohistochemistry in fixed tissues, antigen retrieval is critical:

When performing IHC on human placenta, researchers have successfully used LOX-1/OLR1 antibody (AF1798) at 1 μg/mL with overnight incubation at 4°C, followed by HRP-DAB detection system . Specific staining was observed in cytotrophoblasts.

For paraffin-embedded sections, consider following the protocol outlined in the references, which includes:

• Immersion fixation of tissue

• Paraffin embedding

• Sectioning

• Antigen retrieval with appropriate buffer

• Overnight incubation with primary antibody at 4°C

• Detection using an HRP-DAB system

• Counterstaining with hematoxylin

How can LOX-1/OLR1 antibodies be validated using knockdown and overexpression strategies?

For conclusive validation of antibody specificity, establish control systems using genetic manipulation:

• Overexpression validation:

  • Generate stable cell lines overexpressing LOX-1 (multiple clones)

  • Confirm overexpression by Western blot and quantitative PCR

  • Use as positive controls for antibody validation

• Knockdown validation:

  • Develop stable cell lines expressing shRNA targeting OLR1

  • Test multiple shRNA constructs (e.g., shRNA/LOX-1(A) and shRNA/LOX-1(B))

  • Verify knockdown efficiency using Western blot and qPCR

  • Statistical analysis using one-way ANOVA with appropriate post-tests

This dual validation approach provides strong evidence for antibody specificity and can help resolve contradictory experimental results.

What is the functional significance of LOX-1/OLR1 in cardiovascular and immune research?

LOX-1/OLR1 serves as a dual-function receptor with important roles in both cardiovascular pathology and immune regulation:

• Cardiovascular function: LOX-1 acts as a homodimeric receptor mediating the recognition, internalization, and degradation of oxidatively modified low-density lipoprotein (oxLDL), contributing to atherosclerosis development .

• Immune function: LOX-1 on dendritic cells serves as:

  • A bridge between dendritic cells and B cells to promote humoral immune responses

  • An enhancer of antigen-specific IFNγ-producing CD4+ T cell responses

  • A unique inducer of BAFF and APRIL production, which promote immunoglobulin class switching (particularly IgA1 and IgA2)

Researchers should note that unlike other lectins (Dectin-1, DC-ASGPR, DCIR, DC-SIGN, etc.), only LOX-1 activation induces dendritic cells to produce BAFF and APRIL, making it a specialized target for enhancing both cellular and humoral immunity .

How does LOX-1/OLR1 expression vary across different cell types and conditions?

Understanding LOX-1 expression patterns is essential for experimental design:

Expression levels can be modulated by:

• Inflammatory stimuli

• Interaction with ligands (oxidized-LDL, C-reactive protein, fibronectin)

• Proteolytic cleavage at the neck domain (forming soluble LOX-1)

These expression patterns should inform cell type selection and experimental controls in LOX-1 research.

What are the common issues when detecting LOX-1/OLR1 in Western blot applications?

When troubleshooting Western blot detection of LOX-1/OLR1:

• Molecular weight discrepancy: Expect bands at 40-55 kDa rather than the calculated 31 kDa due to post-translational modifications .

• Sample preparation: For optimal results with antibody 11837-1-AP:

  • Use human liver tissue or bEnd.3 cells as positive controls

  • Store antibody at -20°C (stable for one year after shipment)

  • No aliquoting necessary for -20°C storage

  • Note that 20μl sizes contain 0.1% BSA

• Dilution optimization: The recommended range is 1:1000-1:6000, but titration is necessary for each specific experimental system .

• Validation strategy: Include both positive controls (verified expressing samples) and negative controls (knockdown samples) to confirm specificity.

How should researchers optimize immunohistochemistry protocols for LOX-1/OLR1 detection?

For optimal IHC results with LOX-1/OLR1 antibodies:

• Antigen retrieval optimization:

  • Primary recommendation: TE buffer (pH 9.0)

  • Alternative: Citrate buffer (pH 6.0)

• Dilution optimization:

  • Start with recommended range (1:50-1:500)

  • Test multiple dilutions to identify optimal signal-to-noise ratio

• Positive control selection:

  • Human heart tissue has been validated for IHC

  • Human placenta (cytotrophoblasts specifically) has shown positive results

• Incubation conditions:

  • Consider overnight incubation at 4°C for optimal results

  • Use appropriate detection systems (e.g., HRP-DAB) with validated protocols

• Counterstaining:

  • Hematoxylin provides good contrast for LOX-1/OLR1 DAB staining

What considerations are important for immunoprecipitation experiments with LOX-1/OLR1 antibodies?

For successful immunoprecipitation of LOX-1/OLR1:

• Antibody amount: Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

• Validated cell types: L02 cells have been validated for IP applications

• Co-immunoprecipitation applications: Several publications have validated specific LOX-1 antibodies for co-IP experiments, demonstrating their ability to maintain protein-protein interactions

• Controls:

  • Include IgG control to identify non-specific binding

  • Consider input samples (pre-IP lysate) to confirm target protein presence

  • If available, use LOX-1 knockdown samples as negative controls

• Detection method: After IP, Western blot using a LOX-1 antibody recognizing a different epitope can provide additional validation of specificity

How can LOX-1/OLR1 antibodies be utilized in immunotherapy research?

The unique properties of LOX-1 make it a promising target for therapeutic research:

• Vaccine development: LOX-1's ability to enhance both cellular (Th1) and humoral immune responses makes it a potential target for vaccine strategies, particularly against:

  • Cancer

  • Viral infections

  • Mucosal infections (leveraging its ability to promote IgA1/IgA2 class switching)

• DC-targeted therapy: Antigen targeting to DCs via LOX-1 efficiently elicits antigen-specific IFNγ-producing CD4+ T cell responses, suggesting potential applications in immunotherapy approaches .

• Autoimmunity considerations: Researchers must be aware that LOX-1 activation by oxidized-LDL can induce DCs to secrete BAFF and APRIL, potentially contributing to autoimmune diseases like lupus .

When designing immunotherapy studies targeting LOX-1, researchers should leverage specialized antibodies and consider both the beneficial immune-enhancing properties and potential autoimmune complications.