CD14 PAT87H7AT Antibody

What is CD14 PAT87H7AT Antibody and what is its target protein?

The CD14 PAT87H7AT antibody is a mouse anti-human monoclonal antibody that specifically targets CD14, a 53-55 kDa glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein. This antibody is derived from the hybridization of mouse F0 myeloma cells with spleen cells from BALB/c mice that were immunized with a recombinant human CD14 protein . The antibody recognizes human CD14, which functions primarily as a lipopolysaccharide (LPS) receptor and is prominently expressed on monocytes and macrophages, with lower expression on neutrophils. The target protein CD14 serves as a critical pattern recognition receptor in the innate immune system, facilitating the recognition and clearance of Gram-negative bacteria.

What are the key structural characteristics of CD14 relevant to antibody binding?

CD14 exhibits a horseshoe-like structure typical of leucine-rich-repeat-containing proteins, featuring a hydrophobic pocket at the NH2-terminal region with positively charged residues at the rim that accommodate acylated ligands such as phosphorylated lipid A . Human CD14 contains 375 amino acid residues, including a 19-amino acid signal peptide and a C-terminal hydrophobic region characteristic of GPI-anchored proteins . The protein possesses four potential N-linked glycosylation sites and bears O-linked carbohydrates as well . The PAT87H7AT antibody specifically recognizes epitopes within the recombinant human CD14 protein spanning amino acids 20-349, purified from E. coli . Understanding these structural features is essential for interpreting antibody binding characteristics and potential cross-reactivity in experimental applications.

How does CD14 participate in bacterial recognition and immune signaling?

CD14 forms a multi-receptor complex with Toll-like receptor 4 (TLR4) and MD-2 on the cell membrane to recognize LPS from Gram-negative bacteria . This receptor complex functions through multiple mechanisms:

• CD14 binds LPS at picomolar concentrations and transfers it to the TLR4-MD2 complex to initiate signal transduction

• It enhances cellular sensitivity to low concentrations of LPS, as demonstrated by the markedly reduced responsiveness of CD14-deficient macrophages

• CD14 facilitates the activation of both the MyD88-dependent pathway and the TRAM-TRIF-dependent pathway, leading to pro-inflammatory cytokine production and type I interferon responses, respectively

• Beyond TLR4, CD14 serves as a co-receptor for multiple TLRs (TLR1, 2, 3, 6, 7, and 9), contributing to their ligand recognition capabilities

Additionally, CD14 has been shown to recognize and bind apoptotic cells, suggesting a role in clearing cellular debris during tissue homeostasis .

What experimental techniques can CD14 PAT87H7AT Antibody be applied to?

The CD14 PAT87H7AT antibody has been validated for several experimental applications, making it versatile for CD14 research:

*While the PAT87H7AT clone specifically has been tested for ELISA and Western blot , CD14 antibodies are commonly used in flow cytometry for detecting monocytes and macrophages .

Each application should be properly titrated to determine optimal antibody concentration for specific experimental conditions.

What are the optimal storage and handling conditions for maintaining antibody efficacy?

To maintain optimal activity of the CD14 PAT87H7AT antibody, researchers should adhere to the following storage and handling recommendations:

• For short-term storage (up to 1 month), store at 4°C

• For long-term storage, maintain at -20°C

• Avoid repeated freeze-thaw cycles, which can denature the antibody and reduce its efficacy

• The antibody formulation (1mg/ml containing PBS, pH-7.4, 10% Glycerol and 0.02% Sodium Azide) is designed to maintain stability under proper storage conditions

• Shelf life is approximately 12 months at -20°C and 1 month at 4°C

When working with PE-conjugated CD14 antibodies (though not specifically the PAT87H7AT clone), additional precautions include protecting from light and avoiding freezing to preserve the fluorophore integrity .

How can researchers validate CD14 PAT87H7AT Antibody specificity in their experimental systems?

Validating antibody specificity is crucial for generating reliable experimental data. Researchers should implement the following approaches:

• Positive controls: Use cell lines known to express high levels of CD14 (e.g., THP-1 cells, primary monocytes) to confirm antibody binding

• Negative controls: Test antibody reactivity on CD14-negative cell lines or CD14 knockout cells

• Isotype controls: Include appropriate mouse IgG2b isotype control antibodies at matching concentrations to assess non-specific binding

• Blocking experiments: Pre-incubate the antibody with recombinant CD14 protein before application to demonstrate binding specificity

• Secondary antibody-only controls: Verify the absence of non-specific secondary antibody binding

• Cross-species reactivity: Note that human CD14 shares approximately 65% sequence identity with mouse, rat, rabbit, and bovine homologs , which may affect cross-reactivity studies

Researchers should document these validation steps in their methodology sections to strengthen the reliability of their findings.

How can CD14 PAT87H7AT Antibody be used to investigate TLR4-dependent and TLR4-independent signaling?

CD14 participates in both TLR4-dependent and TLR4-independent signaling pathways, offering multiple research avenues:

For TLR4-dependent signaling studies:

• Use the antibody to block CD14-mediated LPS binding and transfer to TLR4/MD-2, enabling the assessment of specific CD14 contributions to TLR4 activation

• Employ the antibody in co-immunoprecipitation experiments to isolate and characterize CD14-TLR4-MD2 complexes

• Combine CD14 PAT87H7AT antibody with TLR4 pathway inhibitors to delineate the sequence of molecular events following LPS recognition

For TLR4-independent signaling studies:

• Investigate CD14's role in activating the NFAT transcription factor family in myeloid cells, which occurs independently of TLR4

• Use the antibody to track CD14 localization during the activation of alternative signaling pathways

• Combine with TLR4 knockout or TLR4-inhibited systems to isolate CD14-specific effects

The antibody can be particularly valuable in dissecting the mechanisms through which CD14 allows activation of the TLR4-TRAM-TRIF pathway upon LPS stimulation versus TLR4-independent pathways .

What are the considerations when using CD14 PAT87H7AT Antibody to distinguish between membrane-bound and soluble CD14?

CD14 exists in both membrane-bound (mCD14) and soluble (sCD14) forms, which have distinct biological functions. Researchers should consider:

• Experimental design: To detect mCD14, use intact cell assays (flow cytometry, immunofluorescence). For sCD14, use cell-free assays (ELISA, immunoblotting of cell culture supernatants or body fluids)

• Differential functions: mCD14 enhances cellular responses to LPS, while sCD14 can either potentiate LPS responses in CD14-negative cells or inhibit LPS-mediated responses at high concentrations

• Release mechanism: sCD14 can be generated by enzymatic cleavage using phosphatidylinositol-specific phospholipase C , which can be experimentally manipulated to study this conversion

• Quantitative analysis: Establishing standard curves with recombinant sCD14 is essential for accurate quantification in ELISA applications

Since CD14 PAT87H7AT antibody recognizes an epitope in the protein portion of CD14 (amino acids 20-349) , it should theoretically detect both mCD14 and sCD14 forms, making it valuable for comparative studies.

How does CD14's interaction with the lipopolysaccharide binding protein (LBP) affect experimental design?

The interaction between CD14 and LBP is critical for efficient LPS recognition and cellular activation:

• LBP catalyzes the binding of CD14 with LPS, enhancing the sensitivity of the system to low LPS concentrations

• When designing experiments to study CD14-mediated responses to LPS, researchers should consider:

  • Including physiological concentrations of LBP in in vitro systems

  • Controlling for endogenous LBP in serum-containing media

  • Using defined concentrations of recombinant LBP for mechanistic studies

• The CD14 PAT87H7AT antibody can be used in competition assays to determine whether it interferes with the CD14-LBP-LPS interaction

• For co-localization studies, combining CD14 PAT87H7AT with labeled LBP and LPS can help visualize the formation and trafficking of the recognition complex

Understanding this interaction is essential for accurately interpreting data related to CD14's role in bacterial recognition and downstream signaling events.

What are common technical challenges when using CD14 PAT87H7AT Antibody and how can they be addressed?

Researchers may encounter several challenges when working with this antibody:

Since CD14 PAT87H7AT antibody is recommended at a starting dilution of 1:100 , optimization through titration for each specific application and cell type is advisable to address these challenges.

How can researchers address variability in CD14 detection across different tissue samples?

Variability in CD14 detection can stem from biological and technical factors:

• Biological variability management:

  • Normalize CD14 expression to appropriate housekeeping genes/proteins for each tissue type

  • Account for tissue-specific CD14 expression patterns (high in liver and lung, lower in other tissues)

  • Consider disease state effects on CD14 expression (often upregulated during inflammation)

• Technical variability reduction:

  • Standardize sample collection, processing, and storage procedures

  • Process comparative samples simultaneously when possible

  • Include internal reference standards in each experiment

  • Maintain consistent antibody lots when performing longitudinal studies

• Quantification approaches:

  • Use digital image analysis for immunohistochemistry to obtain objective measurements

  • Implement appropriate statistical tests to determine if variability exceeds expected biological variation

CD14 expression can vary significantly based on inflammatory status and tissue microenvironment, requiring careful experimental design and appropriate controls.

How can CD14 PAT87H7AT Antibody contribute to research on CD14's role in metabolic diseases?

Recent research has implicated CD14 in metabolic regulation and obesity-related conditions . The CD14 PAT87H7AT antibody can support this emerging research area through:

• Mechanistic studies:

  • Investigating CD14 expression changes in adipose tissue during obesity development

  • Exploring CD14's role in macrophage polarization in metabolic tissues

  • Examining how CD14-mediated signaling affects insulin sensitivity

• Methodological approaches:

  • Immunophenotyping of metabolic tissue-resident macrophages using flow cytometry or immunohistochemistry

  • Blocking CD14 function in ex vivo adipose tissue cultures to assess metabolic consequences

  • Co-localization studies with metabolic hormone receptors and inflammatory mediators

• Translational applications:

  • Correlation of tissue CD14 levels with metabolic parameters in patient samples

  • Development of prognostic markers based on CD14 expression patterns

  • Target validation for metabolic disease interventions

This application represents an exciting frontier in CD14 research beyond its classic role in infectious disease immunity .

What experimental designs are optimal for studying CD14's role in recognizing and binding apoptotic cells?

CD14 has been shown to bind apoptotic cells , suggesting a role in efferocytosis (clearance of dead cells). Researchers can design experiments to investigate this function:

• In vitro binding assays:

  • Use fluorescently labeled apoptotic cells with CD14-expressing cells in the presence or absence of blocking CD14 PAT87H7AT antibody

  • Perform confocal microscopy to visualize CD14 clustering around apoptotic cell contacts

  • Quantify binding efficiency using flow cytometry before and after CD14 blockade

• Competition experiments:

  • Determine whether LPS and apoptotic cells compete for CD14 binding

  • Investigate whether different forms of cell death produce distinct CD14-binding patterns

  • Assess the contribution of phosphatidylserine exposure to CD14 recognition

• Signaling pathway analysis:

  • Compare CD14-mediated signaling cascades triggered by LPS versus apoptotic cells

  • Investigate the requirement for co-receptors in apoptotic cell recognition

  • Determine the immunological outcomes (pro- vs. anti-inflammatory) of CD14-mediated apoptotic cell recognition

This research direction could provide insights into CD14's dual role in both promoting inflammation during infection and potentially contributing to inflammation resolution through efferocytosis.

How can researchers investigate the structural basis of CD14's interaction with different ligands?

Understanding the structural determinants of CD14's versatile ligand recognition capabilities can be approached using:

• Epitope mapping studies:

  • Use CD14 PAT87H7AT antibody in competition assays with different ligands to determine overlapping binding regions

  • Generate CD14 deletion mutants to identify critical domains for different ligand interactions

  • Employ peptide arrays to identify specific CD14 sequences involved in ligand recognition

• Structural biology approaches:

  • Combine CD14 crystallographic data with molecular modeling to predict ligand binding sites

  • Utilize surface plasmon resonance with recombinant CD14 and various ligands to determine binding kinetics

  • Apply hydrogen-deuterium exchange mass spectrometry to identify conformational changes upon ligand binding

• Comparative species analysis:

  • Leverage the 65% sequence identity between human CD14 and mouse, rat, rabbit, and bovine homologs to identify conserved recognition elements

  • Perform site-directed mutagenesis of divergent residues to assess their contribution to species-specific ligand preferences

  • Create chimeric CD14 proteins to isolate domain-specific functions

These approaches can help elucidate how one receptor can recognize such diverse ligands as LPS, apoptotic cells, and other microbial components, potentially revealing new therapeutic targets.