PGLYRP1 Antibody, FITC conjugated

What is PGLYRP1 and what are its primary biological functions?

PGLYRP1 (also known as PGRP-S, PGRP, TNFSF3L, SBBI68, or UNQ639/PRO1269) is an innate immunity protein that serves multiple critical roles in antimicrobial and antitumor defense systems. It functions primarily as a pattern recognition receptor that binds to murein peptidoglycans (PGN) of Gram-positive bacteria, providing bactericidal activity . Beyond this direct antimicrobial role, PGLYRP1 forms an equimolar complex with heat shock protein HSPA1A to induce programmed cell death through both apoptosis and necroptosis in tumor cell lines by activating the TNFR1 receptor on target cell membranes . Additionally, PGLYRP1 forms a complex with the Ca²⁺-binding protein S100A4 that functions as a chemoattractant capable of inducing lymphocyte movement by serving as a ligand for chemotactic receptors CCR5 and CXCR3 present on immune system cells .

What detection methods are compatible with PGLYRP1 antibodies?

PGLYRP1 antibodies, including FITC-conjugated variants, are compatible with multiple detection methodologies in research settings. The antibody is suitable for Western blotting (WB), immunohistochemistry on paraffin-embedded sections (IHC-P), and immunocytochemistry/immunofluorescence (ICC/IF) applications . For optimal detection in fluorescence microscopy applications, FITC-conjugated antibodies provide direct visualization without requiring secondary antibody incubation steps. The standard size of commercially available PGLYRP1 antibody preparations is typically 50μg, with confirmed reactivity against human PGLYRP1 . When using Western blotting, researchers should anticipate detecting a single PGLYRP1 band of approximately 16 kDa in size, as demonstrated in protein extracts from tissues like cornea and bone marrow .

What species reactivity has been confirmed for PGLYRP1 antibodies?

Commercial PGLYRP1 antibodies, including FITC-conjugated variants, demonstrate confirmed reactivity with human and rat PGLYRP1 . Experimental validation has been conducted using recombinant fragment protein within rat Pglyrp1 (amino acid 1 to C-terminus) as immunogen . For researchers working with murine models, it's important to note that PGLYRP1 expression has been successfully detected in mouse cornea and bone marrow using immunoblotting techniques, but species-specific antibody validation should be conducted before application to ensure cross-reactivity .

How should researchers optimize PGN-FITC internalization studies with monocytes?

When designing PGN-FITC internalization studies with monocytes, researchers should consider implementing one of two established experimental approaches. The first approach utilizes PGN-FITC followed by trypan blue quenching of surface FITC to differentiate between internalized and surface-bound complexes . This method enables accurate quantification of internalization while controlling for potential false positives from surface adherence.

For comprehensive receptor neutralization studies, preincubation of monocytes with monoclonal antibodies against CR1 (J3D3), CR3 (TS1/18), and FCGRs (Fab), either individually or in combination, before exposure to NHS-opsonized PGN-FITC provides valuable insights into receptor-mediated uptake mechanisms . Data analysis should include both the frequency of PGN-positive monocytes and FITC intensities in positive cells (typically log-transformed FITC geometric mean fluorescence intensity). When analyzing results, researchers should note that CR1 serves as the primary C3b receptor involved in PGN recognition, with CR1 neutralization reducing PGN-positive monocyte frequency by approximately 19.3%±1.8% .

What controls should be included when validating PGLYRP1 antibody specificity?

Rigorous validation of PGLYRP1 antibody specificity is essential for generating reliable research data. A comprehensive control strategy should include:

• Positive tissue controls: Bone marrow extracts serve as excellent positive controls due to high endogenous PGLYRP1 expression, typically producing a distinct band at approximately 16 kDa in Western blotting applications .

• Negative controls: Where available, researchers should utilize tissue extracts from PGLYRP1-deficient (Pglyrp1−/−) animals, which should yield no detectable signal in the anticipated 16 kDa region .

• Isotype controls: For flow cytometry and immunofluorescence applications, appropriate isotype controls (such as mIgG1k for many monoclonal antibodies) should be included to establish background staining levels .

• Expression validation: Complementing protein detection with RT-PCR validation is recommended to confirm transcript expression levels. Sample CT values for Pglyrp1 typically range around 23.60±0.02, which can be compared to housekeeping genes like Gapdh (CT ≈ 24.69±0.14) to calculate relative expression levels (2−ΔCT ≈ 2.13±0.19) .

How can researchers effectively quantify PGLYRP1 antibody-mediated detection in flow cytometry?

For accurate quantification of PGLYRP1 antibody-mediated detection in flow cytometry, researchers should implement a standardized analytical approach. Data collection should include both frequency measures (percentage of positive cells within defined populations) and intensity measures (geometric mean fluorescence intensity, preferably log-transformed for statistical analysis) .

When comparing experimental conditions, normalization to isotype-treated controls is recommended, with results presented as percent change from baseline. Statistical analysis should employ appropriate tests for repeated measures, such as RM one-way ANOVA with Holm-Šídák correction for multiple comparisons when data follow normal distribution, or non-parametric alternatives like Friedman's test and Wilcoxon's signed-rank test when normality cannot be assumed .

For experiments involving receptor blocking or competitive binding, presenting data as both raw values and normalized percent inhibition provides comprehensive interpretation. Potential synergistic effects should be evaluated by comparing observed combined inhibition against calculated additive effects, with statistical significance assessed through paired two-tailed t-tests .

How can recombinant PGLYRP1 be effectively produced for antimicrobial research?

For researchers requiring purified PGLYRP1 for antimicrobial assays, several expression systems have been validated with varying yields and activity profiles. The most effective approach involves cloning the human Pglyrp1 gene for subsequent expression in human cell lines such as Expi293F . To overcome yield limitations in stable cell lines, a fusion protein strategy can be implemented by linking PGLYRP1 with IgG light chains, separated by TEV protease sites for subsequent cleavage and purification .

Using this optimized protocol, researchers can achieve yields of approximately 50 mg of purified PGLYRP1 per liter of culture medium . Functional validation of produced recombinant PGLYRP1 should include minimum inhibitory concentration (MIC) determination against reference bacterial strains, with expected MIC values of approximately 12.5 ng/ml against Escherichia coli and Bacillus subtilis . The homologous expression system in human cells ensures appropriate post-translational modifications and preserves antimicrobial activity.

What methodologies can detect PGLYRP1 interaction with bacterial pathogens?

Several methodologies have been validated for investigating PGLYRP1 interaction with bacterial pathogens. For direct binding studies, researchers can analyze sediment and supernatant fractions after centrifugation of PGLYRP1-bacterial mixtures by Western blotting with anti-6His antibodies . In this approach, PGLYRP1 is predominantly detected in the sediment fraction after centrifugation of the mixture, confirming attachment to bacterial elementary bodies .

For functional studies, MIC determination protocols provide quantitative assessment of antimicrobial activity. The significantly higher MICs observed against intracellular pathogens like Chlamydia trachomatis (200 ng/ml) compared to free-living bacteria such as E. coli (12.5 ng/ml) reflect pathogen-specific resistance mechanisms related to their lifecycle . Beyond MIC determination, researchers can evaluate the impact on infectious progeny production by comparing inclusion-forming units (IFUs) after PGLYRP1 exposure, which demonstrates the ability of PGLYRP1 to reduce the infectious capability of bacterial elementary bodies in subsequent infection cycles .

How does PGLYRP1 activity compare with other antimicrobial peptides against intracellular pathogens?

PGLYRP1 demonstrates distinctive antimicrobial activity against intracellular pathogens compared to other antimicrobial peptides. While several host immune effectors including defensins, cathelicidin peptide LL-37, and protegrin have demonstrated antichlamydial effects, PGLYRP1 exhibits a unique mechanism of action . The MIC values for PGLYRP1 against Chlamydia trachomatis (approximately 200 ng/ml) are notably higher than those observed against extracellular bacteria (12.5 ng/ml for E. coli and B. subtilis), suggesting specialized adaptation to the intracellular pathogen's lifestyle .

Mechanistically, PGLYRP1's activity against intracellular pathogens involves direct attachment to elementary bodies, which activates the chlamydial two-component stress response system . This activation leads to developmental inhibition rather than immediate bacterial lysis, differentiating PGLYRP1's mode of action from many classical antimicrobial peptides. For comparative studies with other antimicrobial peptides, researchers should implement standardized infection models using HeLa cells and calculate inhibition percentages at equivalent molar concentrations rather than mass-based dosing to account for molecular weight differences .

What are the common challenges in FITC-conjugated antibody applications and how can they be addressed?

When working with FITC-conjugated PGLYRP1 antibodies, researchers may encounter several technical challenges that can impact experimental outcomes. Photobleaching represents a significant limitation of FITC fluorophores during extended imaging sessions or when using high-intensity excitation sources. To mitigate this issue, researchers should minimize sample exposure to excitation light during preparation and imaging, utilize antifade mounting media containing appropriate preservatives, and consider alternative workflows such as capturing images from unexposed fields when sequential imaging is required.

Another common challenge is the relatively low fluorescence quantum yield and pH sensitivity of FITC compared to newer generation fluorophores. Maintaining consistent buffer pH (ideally above pH 7.0) throughout experimental procedures is essential for reproducible signal intensity. For applications requiring maximum sensitivity, particularly when detecting low-abundance PGLYRP1 expression, signal amplification systems or alternative conjugates with higher quantum yields may be preferable.

How can researchers optimize PGLYRP1 detection in complex tissue samples?

For optimal PGLYRP1 detection in complex tissue samples, several tissue-specific optimization strategies should be considered. First, appropriate antigen retrieval methods must be selected based on tissue fixation protocols. For formalin-fixed, paraffin-embedded samples, heat-induced epitope retrieval using citrate buffer (pH 6.0) provides effective recovery of PGLYRP1 epitopes while maintaining tissue morphology .

Signal-to-noise optimization requires careful antibody titration specific to each tissue type, with empirical determination of optimal concentration. Starting dilutions of 1:100 to 1:500 are recommended, with subsequent adjustment based on specific signal intensity relative to background. For tissues with high autofluorescence (such as skin or brain), implementing quenching protocols with Sudan Black B or spectral unmixing during image acquisition significantly improves detection specificity .

For dual or multi-color applications, careful selection of complementary fluorophores is essential to minimize spectral overlap with FITC. Combinations with far-red fluorophores such as Cy5 or Alexa Fluor 647 for secondary targets provide excellent spectral separation, while combinations with PE or Texas Red require appropriate compensation controls.

What quantitative analytical approaches are recommended for PGLYRP1 expression studies?

For rigorous quantitative analysis of PGLYRP1 expression, researchers should implement a multi-platform approach combining protein and transcript quantification. At the transcript level, quantitative RT-PCR using validated primers enables sensitive detection of Pglyrp1 expression. Expected CT values in expressing tissues typically range around 23.60±0.02, which should be normalized to stable reference genes such as Gapdh (CT ≈ 24.69±0.14) . Relative expression calculation using the 2−ΔCT method provides standardized quantification with expected values of approximately 2.13±0.19 in PGLYRP1-expressing tissues .

For protein-level quantification in flow cytometry applications, geometric mean fluorescence intensity (gMFI) provides the most appropriate metric, ideally log-transformed for statistical analysis . When analyzing changes in PGLYRP1-positive cell populations, both frequency measures and intensity distributions should be reported to capture population heterogeneity. For Western blotting quantification, normalization to stable loading controls followed by densitometric analysis enables semi-quantitative comparison between samples, with expected PGLYRP1 band intensity at approximately 16 kDa .

How is PGLYRP1 being explored in non-classical immune cell populations?

Recent research has expanded the investigation of PGLYRP1 beyond its traditionally studied expression in neutrophils and bone marrow. A significant emerging direction involves the identification and characterization of PGLYRP1 in non-classical immune cell populations and epithelial tissues. For instance, PGLYRP1 has been identified as a novel antimicrobial protein in the corneal epithelium, where it plays a protective role against bacterial infections at the ocular surface . This discovery, facilitated by genomewide differential gene expression analysis, reveals previously unrecognized tissue-specific immune functions of PGLYRP1.

Researchers exploring PGLYRP1 in non-classical locations should implement a sequential validation approach beginning with transcript detection through RT-PCR, followed by protein validation using Western blotting with appropriate positive controls (bone marrow) and negative controls (tissues from Pglyrp1−/− animals) . Functional studies in these newly identified PGLYRP1-expressing tissues should assess antimicrobial activity against tissue-relevant pathogens using bacterial killing assays with clinical isolates relevant to the tissue microenvironment.

What is the potential of PGLYRP1 as a therapeutic target for infectious diseases?

The antimicrobial properties of PGLYRP1 present compelling opportunities for therapeutic development against challenging infectious diseases. Particularly promising is PGLYRP1's demonstrated activity against intracellular pathogens like Chlamydia trachomatis, with defined MIC values (200 ng/ml) providing quantitative therapeutic targets . The attachment of PGLYRP1 to chlamydial elementary bodies and subsequent activation of stress response systems suggests potential for treating persistent infections that resist conventional antibiotics .

For researchers exploring therapeutic applications, recombinant PGLYRP1 production protocols yielding 50 mg/liter in Expi293F human cell lines provide scalable sources of functional protein . Therapeutic development strategies should focus on stability optimization, targeted delivery systems, and combination approaches with conventional antibiotics. Evaluation metrics should include not only direct antimicrobial activity but also effects on infectious progeny production, which demonstrates PGLYRP1's ability to break transmission cycles by reducing elementary body infectivity in subsequent generations .

How does PGLYRP1 function in the broader context of complement-mediated immunity?

The interaction between PGLYRP1 and the complement system represents a complex and understudied aspect of innate immunity integration. Research indicates that complement receptors play significant roles in PGLYRP1-mediated recognition and cellular responses. Specifically, CR1 functions as the main C3b receptor involved in peptidoglycan recognition, with CR1 neutralization reducing PGN-positive monocyte frequency by approximately 19.3%±1.8% . Interestingly, CR1 and CR3 demonstrate overlapping but distinct roles, with CR1 primarily mediating capture while both receptors influence uptake quantity .

For researchers investigating these interactions, receptor neutralization studies using monoclonal antibodies against CR1 (J3D3) and CR3 (TS1/18) provide valuable mechanistic insights . Quantitative assessment should include both frequency measures of PGN-positive cells and intensity metrics within positive populations, as these parameters may be differentially affected by receptor inhibition . Combined inhibition studies reveal potential synergistic effects between complement receptors, with observed combined inhibition (58.2%±3.2%) differing significantly from calculated additive effects (71.6%±6.6%), suggesting complex receptor interactions worthy of further investigation .