PLGRKT Antibody

What is PLGRKT and why is it important in research?

PLGRKT (plasminogen receptor with a C-terminal lysine) is a transmembrane protein with unique structural characteristics, having both N- and C-terminal domains exposed on the extracellular face of the cell. The C-terminal lysine functions to tether plasminogen to cell surfaces. This protein is critical in the plasminogen activation system, which plays roles in various physiological and pathological processes. PLGRKT is broadly expressed in cells and tissues throughout organisms, and its sequence is remarkably conserved phylogenetically. Research has identified its necessity for lactation and species survival, making it a protein of significant biological importance .

What are the basic properties of human PLGRKT?

In humans, the canonical PLGRKT protein has a length of 147 amino acid residues and a molecular weight of approximately 17.2 kDa. Its primary subcellular localization is in the cell membrane, where it functions as a receptor for plasminogen. PLGRKT is predominantly expressed in peripheral blood cells and monocytes. The protein is also known by several synonyms, including 5033414D02Rik, transmembrane protein C9orf46, and plasminogen receptor (KT) .

What are the primary applications for PLGRKT antibodies in research?

PLGRKT antibodies are employed in multiple immunodetection techniques to study the expression, localization, and function of this plasminogen receptor. The most common applications include:

These techniques allow researchers to investigate PLGRKT expression patterns and their correlation with biological processes such as inflammation and cellular migration .

How should I select the most appropriate PLGRKT antibody for my research?

When selecting a PLGRKT antibody, consider these research-critical factors:

• Epitope location: Determine whether you need an antibody targeting the N-terminal, internal, or C-terminal region of PLGRKT. This is particularly important since PLGRKT has both terminals exposed on the cell surface.

• Species cross-reactivity: Verify the antibody's reactivity with your experimental model. Many PLGRKT antibodies show cross-reactivity with human, mouse, and rat proteins, but specificities vary among commercial products .

• Validated applications: Confirm that the antibody has been validated for your specific application (Western blot, ELISA, immunofluorescence, etc.) through published literature or manufacturer testing.

• Monoclonal vs. polyclonal: Consider the trade-offs between specificity (monoclonal) and sensitivity (polyclonal) based on your experimental needs. For precise localization studies, monoclonal antibodies may be preferable .

• Clone information: For monoclonal antibodies, review the specific clone's performance in applications similar to yours through literature searches.

What controls should I include when working with PLGRKT antibodies?

Proper experimental controls are essential for reliable results:

• Positive control: Include samples known to express PLGRKT, such as peripheral blood monocytes or CD14++CD16+ human monocytes, which express high levels of PLGRKT .

• Negative control: Use PLGRKT-knockout cells/tissues or samples known not to express the protein.

• Isotype control: Include an appropriate isotype control antibody to identify non-specific binding.

• Blocking peptide control: Use a competitive inhibition approach with the immunizing peptide to confirm specificity.

• Secondary antibody-only control: Perform staining with only the secondary antibody to identify background signal.

• siRNA knockdown: For advanced validation, use PLGRKT siRNA knockdown samples to confirm antibody specificity.

What are the optimal conditions for detection of PLGRKT in Western blotting?

For optimal PLGRKT detection via Western blot:

• Sample preparation: Use cell membrane fractionation techniques to enrich for PLGRKT, as it is a transmembrane protein.

• Protein denaturation: Employ reducing conditions, but avoid excessive heating that might cause aggregation of membrane proteins.

• Gel percentage: Use 12-15% SDS-PAGE gels to effectively resolve the 17.2 kDa PLGRKT protein.

• Transfer conditions: Optimize transfer of small proteins using higher methanol concentrations in the transfer buffer.

• Blocking: A 5% BSA blocking solution often provides better results than milk for detecting membrane proteins.

• Antibody dilution: Start with manufacturer-recommended dilutions (typically 1:500 to 1:1000) and optimize as needed.

• Detection system: Choose a detection system with appropriate sensitivity for detecting PLGRKT's relatively low abundance in many cell types .

How does PLGRKT expression vary across monocyte and macrophage subsets?

PLGRKT expression varies significantly across immune cell subsets, with important functional implications:

• Monocyte subsets: Proinflammatory CD14++CD16+ human monocytes and Ly6Chigh mouse monocytes express the highest levels of PLGRKT compared to other subsets. This differential expression correlates with their enhanced capacity to bind plasminogen .

• Macrophage polarization: Proinflammatory macrophages (polarized with LPS and IFN-γ) show significantly higher expression of PLGRKT compared to alternatively activated macrophages (polarized with IL-4 and IL-13) .

• Neutrophils: Neutrophils express relatively low levels of PLGRKT compared to monocytes and macrophages, correlating with their lower plasminogen binding capacity .

This differential expression pattern suggests a specialized role for PLGRKT in inflammatory processes, particularly in the function of proinflammatory monocyte and macrophage populations.

What methodologies can accurately quantify PLGRKT expression differences between cell types?

To accurately quantify PLGRKT expression differences:

• Flow cytometry: Use anti-PLGRKT antibodies conjugated with fluorophores or with appropriate secondary antibodies to measure surface expression levels. This method allows for simultaneous analysis of PLGRKT expression alongside other cell surface markers to identify specific subpopulations .

• Quantitative RT-PCR: Measure PLGRKT mRNA levels in sorted cell populations or polarized macrophages. This approach provides information about transcriptional regulation .

• Immunofluorescence microscopy: Visualize PLGRKT distribution on different cell types using fluorescently labeled antibodies. Combine with markers for cell identification, such as CD80 for proinflammatory macrophages .

• Western blotting: Quantify protein expression in lysates from purified cell populations, with normalization to appropriate loading controls.

• Mass cytometry: For advanced analyses, use CyTOF (cytometry by time-of-flight) to simultaneously measure PLGRKT alongside dozens of other markers to comprehensively profile immune cell subsets.

How can I investigate the role of PLGRKT in cell migration?

To investigate PLGRKT's role in cell migration:

• Transwell migration assays: Use Transwell inserts (5.0-μm pore size) to measure directed migration of cells with varying PLGRKT expression levels. Compare migration in the presence or absence of anti-PLGRKT monoclonal antibodies to determine PLGRKT-dependent effects .

• Plasminogen activation inhibitors: Include ε-amino-caproic acid (a lysine analog that blocks plasminogen binding) or aprotinin (a plasmin inhibitor) to assess plasmin dependence of migration .

• PLGRKT knockdown/knockout approaches: Use siRNA, CRISPR-Cas9, or cells from PLGRKT knockout mice to directly assess how PLGRKT deficiency affects migration capacity.

• Live-cell imaging: Track cell movement in real-time using fluorescently labeled cells with different PLGRKT expression levels.

• In vivo migration models: Use peritonitis models to assess recruitment of different leukocyte populations in wild-type versus PLGRKT-knockout mice, or in the presence of anti-PLGRKT antibodies .

What experimental approaches can evaluate the interaction between PLGRKT and plasminogen?

To study PLGRKT-plasminogen interactions:

• Plasminogen binding assays: Measure the binding of fluorescently-labeled plasminogen to cells expressing different levels of PLGRKT. Flow cytometry can quantify binding differences between cell types or before and after PLGRKT manipulation .

• Co-immunoprecipitation: Precipitate PLGRKT and probe for associated plasminogen, or vice versa, to demonstrate physical interaction.

• Surface plasmon resonance: Measure binding kinetics between purified PLGRKT and plasminogen to determine affinity constants.

• FRET (Förster Resonance Energy Transfer): Evaluate proximity-based interactions between fluorescently labeled PLGRKT and plasminogen on cell surfaces.

• Competitive inhibition studies: Use C-terminal lysine analogs or the anti-PLGRKT monoclonal antibody to competitively inhibit plasminogen binding and assess functional consequences .

How does PLGRKT interact with the urokinase receptor (uPAR) system?

PLGRKT physically associates with the urokinase receptor (uPAR), creating an efficient system for plasminogen activation:

• Co-localization studies: Use dual-color immunofluorescence to visualize the spatial relationship between PLGRKT and uPAR on cell surfaces.

• Proximity ligation assay: Detect protein-protein interactions between PLGRKT and uPAR in situ with single-molecule resolution.

• Functional interaction assays: Measure plasminogen activation in the presence of urokinase-type plasminogen activator (uPA) with cells expressing both, either, or neither receptor.

• Co-immunoprecipitation: Isolate protein complexes containing both PLGRKT and uPAR to confirm physical association.

• Effect of uPAR blockade: Determine how blocking uPAR affects PLGRKT-dependent functions, and vice versa, to understand the interdependence of these systems in cellular processes such as migration .

What is the evidence for PLGRKT's role in inflammatory cell recruitment?

PLGRKT plays a critical role in selective inflammatory cell recruitment:

• Peritonitis models: In thioglycollate-induced peritonitis, PLGRKT knockout mice show significantly impaired macrophage recruitment (76% lower at 72 hours) compared to wild-type littermates, without affecting neutrophil recruitment .

• Antibody blockade effects: Treatment with anti-PLGRKT monoclonal antibodies inhibits both macrophage recruitment (by 53%) and lymphocyte recruitment (by 60%) in peritonitis models, without affecting neutrophil or eosinophil recruitment .

• Cell-type specificity: The selective effect on monocyte/macrophage and lymphocyte recruitment, but not neutrophils, correlates with the differential expression pattern of PLGRKT across leukocyte populations .

• Plasminogen dependence: Similar patterns of impaired macrophage and lymphocyte recruitment, without effects on neutrophils, are observed in plasminogen-deficient mice, supporting the functional relationship between plasminogen and PLGRKT .

• Inflammatory marker expression: Altered levels of inflammatory cytokines, including IL-6 and IL-10, accompany the impaired recruitment of Ly6Chigh monocytes in PLGRKT-deficient mice .

How can researchers distinguish between the roles of PLGRKT in inflammation initiation versus resolution?

To differentiate PLGRKT's roles in inflammation initiation versus resolution:

• Time-course experiments: Monitor PLGRKT expression and function at different phases of the inflammatory response (initiation, peak, resolution) using models like thioglycollate-induced peritonitis .

• Cell-type specific analyses: Separately examine PLGRKT's effects on different cell populations known to be involved in either initiating inflammation (e.g., certain monocyte subsets) or promoting resolution (e.g., specific macrophage phenotypes).

• Cytokine profiling: Measure both pro-inflammatory (TNF-α, IL-1β, IL-6) and resolution-phase (IL-10, TGF-β) cytokines in PLGRKT-manipulated models.

• Conditional knockout models: Use inducible, cell-type specific PLGRKT knockout systems to selectively delete the receptor during different phases of inflammation.

• Resolution indices: Assess established markers of inflammation resolution (reduction in neutrophil numbers, clearance of apoptotic cells, tissue repair markers) in the presence and absence of functional PLGRKT.

What are common technical challenges when using PLGRKT antibodies in flow cytometry?

When using PLGRKT antibodies for flow cytometry:

• Low signal intensity: PLGRKT may have relatively low expression in some cell types. This can be addressed by:

  • Using signal amplification methods like biotin-streptavidin systems

  • Selecting brighter fluorophores (PE, APC) rather than FITC

  • Optimizing antibody concentration and incubation conditions

• Background signal issues: Minimize by:

  • Using proper blocking agents (Fc block, serum matching secondary antibody species)

  • Including FMO (fluorescence minus one) controls

  • Using isotype controls at the same concentration as the primary antibody

• Epitope accessibility: Since PLGRKT is a transmembrane protein, certain epitopes may be difficult to access. Try:

  • Testing antibodies directed against different regions of the protein

  • Using gentle permeabilization methods if targeting intracellular domains

  • Avoiding harsh fixation conditions that might alter protein conformation

• Distinguishing specific binding: Confirm specificity by:

  • Comparing staining in PLGRKT-positive vs. PLGRKT-negative/knockdown cells

  • Using competitive blocking with immunizing peptides

  • Correlating staining with known expression patterns across cell types

How should researchers address nonspecific binding when using PLGRKT antibodies in tissue sections?

To minimize nonspecific binding in immunohistochemistry:

• Optimal fixation: Use gentle fixation methods (2-4% paraformaldehyde) to preserve epitope accessibility while maintaining tissue architecture.

• Comprehensive blocking: Block with:

  • Serum from the same species as the secondary antibody (5-10%)

  • BSA (1-3%) to reduce nonspecific protein interactions

  • Additional blockers for specific tissues (e.g., milk for adipose tissue)

• Antigen retrieval optimization: Test multiple antigen retrieval methods (citrate, EDTA, enzymatic) to determine which works best for PLGRKT detection in your specific tissue.

• Antibody titration: Perform careful antibody dilution series to identify the optimal concentration that maximizes specific signal while minimizing background.

• Multiple controls: Include tissue sections with:

  • Primary antibody omitted

  • Isotype control antibody at matching concentration

  • PLGRKT-deficient tissue (if available)

  • Pre-absorption of antibody with immunizing peptide

• Signal amplification systems: For low-abundance detection, consider tyramide signal amplification or polymer-based detection systems, while carefully controlling for increased background .

What approaches can resolve contradictory results when different PLGRKT antibodies yield inconsistent findings?

When faced with discrepant results using different PLGRKT antibodies:

• Epitope mapping: Determine which regions of PLGRKT each antibody targets. Differences may be due to epitope accessibility or post-translational modifications affecting specific regions.

• Validation using genetic approaches: Confirm antibody specificity using:

  • PLGRKT knockout/knockdown samples as negative controls

  • PLGRKT overexpression systems as positive controls

  • Rescue experiments in knockout systems

• Cross-validation with non-antibody methods: Support findings with:

  • mRNA expression analysis (RT-PCR, in situ hybridization)

  • Tagged PLGRKT constructs detected with anti-tag antibodies

  • Mass spectrometry-based protein detection

• Multimodal approach: Use multiple antibodies targeting different epitopes and multiple detection methods, looking for convergent evidence.

• Literature comparison: Systematically compare your results with published findings, considering methodological differences that might explain discrepancies.

• Functional validation: Ultimately, connect antibody staining patterns with functional outcomes to determine which pattern correlates with biological activity .

How can PLGRKT antibodies be used to study its role in pathological conditions?

PLGRKT antibodies can elucidate this receptor's role in disease through:

• Atherosclerosis research: Immunohistochemical analysis of human carotid plaques reveals high expression of PLGRKT in proinflammatory macrophages within atherosclerotic lesions. Use antibodies to co-stain for PLGRKT alongside markers like CD80 to identify specific macrophage populations in arterial tissue .

• Obesity and adipose tissue inflammation: Adipose tissue samples from obese subjects show differential PLGRKT expression in macrophage populations. Use PLGRKT antibodies to track macrophage infiltration and phenotype changes in adipose tissue during obesity development .

• Cancer metastasis: Given PLGRKT's role in cell migration and plasminogen activation, investigate its expression in tumor cells and tumor-associated macrophages using antibody-based techniques.

• Autoimmune disease: Examine the correlation between PLGRKT expression levels on immune cells and disease severity or progression in conditions like rheumatoid arthritis or multiple sclerosis.

• Therapeutic targeting: Use blocking antibodies against PLGRKT to assess the potential for therapeutic intervention in inflammatory diseases .

What methodological approaches can assess the molecular interactions between PLGRKT and its binding partners?

To investigate PLGRKT's molecular interactions:

• Immunoprecipitation-mass spectrometry: Use anti-PLGRKT antibodies to pull down PLGRKT and its associated proteins, followed by mass spectrometry identification of binding partners.

• Proximity-dependent biotinylation: Employ BioID or APEX2 approaches with PLGRKT fusion proteins to identify proximal proteins in living cells.

• FRET/BRET analysis: Measure energy transfer between fluorescently tagged PLGRKT and potential interaction partners to confirm direct interactions and measure binding dynamics.

• Surface plasmon resonance: Determine binding kinetics and affinity constants for purified PLGRKT and its partners using label-free detection methods.

• Cross-linking mass spectrometry: Identify specific amino acid residues involved in protein-protein interactions through chemical cross-linking followed by mass spectrometry analysis.

• Structural biology approaches: Use X-ray crystallography or cryo-electron microscopy to determine the three-dimensional structure of PLGRKT alone or in complex with binding partners .

How can researchers quantitatively assess the impact of PLGRKT expression levels on plasminogen activation kinetics?

To quantitatively analyze PLGRKT's effect on plasminogen activation: