PGLYRP1 Antibody, Biotin conjugated

What is PGLYRP1 and what biological functions does it serve?

PGLYRP1 (Peptidoglycan Recognition Protein 1), also known as Peptidoglycan Recognition Protein Short (PGRP-S), functions primarily as a pattern recognition receptor in the innate immune system. This protein specifically binds to murein peptidoglycans (PGN) of Gram-positive bacteria, exhibiting bactericidal activity against these organisms. It also demonstrates bacteriostatic activity toward Gram-negative bacteria, highlighting its broad antimicrobial profile .

On a molecular level, PGLYRP1 plays an essential role in antimicrobial humoral immune responses mediated by antimicrobial peptides. It contributes to the killing of foreign organisms and participates in host response to bacterial invasion . Research has further revealed that PGLYRP1 can form complexes with heat shock protein HSPA1A, inducing programmed cell death through apoptosis and necroptosis in tumor cell lines by activating the TNFR1 receptor on target cell membranes .

Additionally, PGLYRP1 (also referred to as Tag7 in some literature) can bind to the receptors TREM-1 and TNFR1, with evidence indicating that its binding to TREM-1 triggers the secretion of proinflammatory cytokines .

How does PGLYRP1 Antibody, Biotin conjugated differ from other PGLYRP1 antibody formats?

The PGLYRP1 Antibody, Biotin conjugated format offers specific methodological advantages for research applications compared to unconjugated or differently labeled antibodies. The biotin conjugation enables this antibody to work efficiently with enzyme-conjugated streptavidin systems, leveraging the strong biotin-streptavidin binding interaction (one of the strongest non-covalent interactions in nature) to enhance detection sensitivity .

This format is particularly useful in multi-step detection protocols like ELISA, where the biotin-conjugated antibody binds to the target PGLYRP1 protein, followed by addition of enzyme-conjugated streptavidin (typically horseradish peroxidase-linked) that binds to the biotin molecules. This ultimately produces a measurable signal when appropriate substrate is added .

Other available conjugates include various fluorophores (AF350, AF405, AF488, AF555, AF594, AF647, AF680, and AF750), each with distinct excitation and emission profiles that make them suitable for different fluorescence-based applications . The choice between biotin and fluorophore conjugation should be guided by your specific experimental methodology, detection system, and desired sensitivity level.

What is the recommended storage protocol for maintaining long-term antibody activity?

To maintain optimal activity of PGLYRP1 Antibody, Biotin conjugated, proper storage conditions are critical. Upon receipt, the antibody should be stored at either -20°C or -80°C . It is important to avoid repeated freeze-thaw cycles as these can lead to protein denaturation and loss of antibody function.

For experimental protocols that require repeated use of the antibody, consider preparing small aliquots of the stock solution prior to freezing. Each aliquot should contain sufficient antibody for a single experiment to minimize freeze-thaw cycles of the main stock.

If working with the antibody over several days, short-term storage at 4°C in the dark is acceptable for certain conjugated formats , but biotin-conjugated antibodies generally maintain better stability when kept frozen between uses. When handling the antibody, minimize exposure to light, particularly for fluorophore-conjugated variants, as photobleaching can reduce signal intensity over time.

What is the optimal protocol for using PGLYRP1 Antibody, Biotin conjugated in ELISA?

For optimal performance in ELISA applications, PGLYRP1 Antibody, Biotin conjugated should be used according to the following methodology:

ELISA Protocol Optimization:

• Antibody Dilution: The recommended dilution range for biotin-conjugated PGLYRP1 antibody is 1:50 to 1:1,000 when working with enzyme-conjugated streptavidin systems . Begin optimization at 1:250 and adjust based on signal-to-noise ratio.

• Sample Preparation: For cell culture supernatants, serum, or plasma samples, centrifuge to remove particulates and use immediately or aliquot and store at -20°C or -80°C.

• Assay Procedure:

  • Add 100 μl of standards or samples to pre-coated microtiter plate wells

  • Add the optimized dilution of biotin-conjugated PGLYRP1 antibody

  • Incubate at 37°C for 60 minutes

  • Wash plate thoroughly (4-6 times) with wash buffer

  • Add 100 μl of HRP-conjugated streptavidin solution

  • Incubate at 37°C for 30 minutes

  • Wash plate thoroughly (4-6 times)

  • Add 100 μl of TMB substrate solution

  • Incubate at 37°C for 10-20 minutes in the dark

  • Add 50 μl of stop solution (1N H₂SO₄)

  • Read absorbance at 450 nm within 30 minutes

• Data Analysis: Generate a standard curve using known concentrations of PGLYRP1 and determine sample concentrations by comparing absorbance values to this curve.

How can PGLYRP1 Antibody, Biotin conjugated be incorporated into immunohistochemistry protocols?

While the provided search results specifically mention ELISA as a tested application for the PGLYRP1 Antibody, Biotin conjugated , adapting it for immunohistochemistry (IHC) requires careful protocol optimization:

IHC Protocol Adaptation:

• Tissue Preparation:

  • Fix tissue sections in 4% paraformaldehyde or 10% neutral buffered formalin

  • Embed in paraffin or prepare frozen sections based on target sensitivity

  • For paraffin sections, perform antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Blocking and Antibody Application:

  • Block endogenous peroxidase with 3% H₂O₂

  • Apply protein block (5% normal serum)

  • Block endogenous biotin using a biotin blocking kit

  • Apply primary antibody against PGLYRP1 (non-conjugated)

  • Apply biotin-conjugated PGLYRP1 antibody at 1:50-1:250 dilution

  • Incubate in a humidified chamber at 4°C overnight

• Detection:

  • Apply HRP-conjugated streptavidin

  • Develop with DAB substrate

  • Counterstain with hematoxylin

  • Mount and analyze under microscope

• Controls:

  • Include negative controls (omitting primary antibody)

  • Include positive controls (tissues known to express PGLYRP1)

  • Consider using PGLYRP1 knockout tissues as specificity controls

When adapting this antibody for IHC, begin with a higher concentration than used for ELISA (approximately 1:50 dilution) and optimize based on signal intensity and background.

What methodological approaches can be used to study PGLYRP1's role in asthma models?

Research has demonstrated that PGLYRP1 plays a significant role in experimental asthma models, with Pglyrp1-deficient mice developing less severe asthma than wild type mice following house dust mite allergen sensitization . To investigate this role using PGLYRP1 Antibody, Biotin conjugated, consider the following methodological approaches:

In Vivo Experimental Design:

• Mouse Model Development:

  • Compare wild-type and Pglyrp1-/- mice

  • Sensitize with house dust mite (HDM) allergen through intranasal administration

  • Assess asthma parameters at different time points (3-5 weeks)

• Assessment Parameters:

  • Bronchial hyperresponsiveness via airway resistance measurements

  • Inflammatory cell infiltration in bronchoalveolar lavage fluid (BALF)

  • Lung histopathology for remodeling features

• Molecular Analysis:

  • Use PGLYRP1 Antibody, Biotin conjugated to detect and quantify PGLYRP1 expression in lung tissues

  • Measure cytokine levels (IL-4, IL-5, IL-17) in lung homogenates

  • Assess IgE levels in BALF, lung homogenates, and serum

Data from PGLYRP1 Knockout Studies:

This experimental approach allows for comprehensive examination of how PGLYRP1 influences asthma pathogenesis through modulation of immune cell populations and inflammatory mediators .

How does PGLYRP1 interact with the TREM-1 receptor and what are the implications for inflammatory regulation?

Recent research has revealed a complex interaction between PGLYRP1 (Tag7) and the TREM-1 receptor, with significant implications for inflammatory regulation. PGLYRP1 binding to TREM-1 has been shown to trigger the secretion of proinflammatory cytokines . This interaction forms part of a sophisticated immune regulation pathway that can be studied using the following methodological approaches:

Investigating PGLYRP1-TREM-1 Interactions:

• Co-immunoprecipitation Studies:

  • Use PGLYRP1 Antibody, Biotin conjugated to pull down PGLYRP1 and associated proteins

  • Detect TREM-1 co-precipitation through Western blotting

  • Perform reciprocal co-IP with TREM-1 antibodies to confirm interaction

• Cell-Based Assays:

  • Treat human monocytes with recombinant PGLYRP1

  • Block TREM-1 with specific antibodies in parallel experiments

  • Measure cytokine production (TNF-α, IL-1β, IL-6) to assess signaling outcomes

• Surface Plasmon Resonance:

  • Determine binding kinetics between purified PGLYRP1 and TREM-1

  • Assess how specific mutations in either protein affect binding affinity

Interestingly, research on PGLYRP1/Tag7 peptides has demonstrated their ability to decrease proinflammatory responses in human blood cells and in a mouse model of diffuse alveolar damage of the lung . This suggests that while full-length PGLYRP1 may promote inflammation through TREM-1 binding, specific peptide fragments might have opposing effects, potentially by acting as competitive inhibitors of the PGLYRP1-TREM-1 interaction.

This dual functionality positions PGLYRP1 as a potential therapeutic target in inflammatory conditions, where modulating its interaction with TREM-1 could help regulate excessive inflammatory responses.

What are the current research findings on PGLYRP1's role in tumor immunology?

PGLYRP1 has emerged as a significant player in tumor immunology through several distinct mechanisms. Understanding these roles requires sophisticated experimental approaches that can leverage PGLYRP1 Antibody, Biotin conjugated:

PGLYRP1 in Tumor Immunity:

• Complex Formation with Heat Shock Proteins:

  • PGLYRP1 forms an equimolar complex with heat shock protein HSPA1A (Hsp70)

  • This complex can induce programmed cell death through both apoptosis and necroptosis in tumor cell lines

  • The mechanism involves activation of the TNFR1 receptor on target cell membranes

• Chemotactic Activity:

  • PGLYRP1 (Tag7) acts in complex with the calcium-binding protein S100A4

  • This complex functions as a chemoattractant capable of inducing lymphocyte movement

  • Mechanistically, it serves as a ligand for chemotactic receptors CCR5 and CXCR3 on immune cells

• Cytotoxic Lymphocyte Activation:

  • PGLYRP1 promotes activation of lymphocytes that can kill virus-infected cells and tumor cells

  • This occurs through modulation of their target-cell specificity

  • The induction of cytotoxicity on monocyte surfaces requires interaction with the TREM1 receptor

Experimental Approaches:

To investigate these mechanisms using PGLYRP1 Antibody, Biotin conjugated, researchers can:

• Perform immunohistochemistry on tumor tissue microarrays to correlate PGLYRP1 expression with clinical outcomes

• Use flow cytometry with biotin-conjugated antibodies to assess PGLYRP1 expression on tumor-infiltrating immune cells

• Develop in vitro co-culture systems to study how PGLYRP1 mediates interactions between immune cells and tumor cells

These findings suggest that PGLYRP1 may have untapped potential as both a biomarker and therapeutic target in cancer immunotherapy. Understanding its complex roles in tumor immunity could lead to novel approaches for enhancing anti-tumor immune responses.

How can researchers investigate the contradictory pro-inflammatory and anti-inflammatory effects of PGLYRP1 in different disease models?

One of the most intriguing aspects of PGLYRP1 biology is its apparently contradictory roles in inflammation, acting as both pro-inflammatory and anti-inflammatory in different contexts. This dichotomy presents a complex research challenge that can be addressed through careful experimental design:

Methodological Approach to Resolving Contradictory Effects:

• Comparative Disease Models:

  • Asthma Model: In this context, Pglyrp1-/- mice develop less severe disease than wild type mice, suggesting a pro-inflammatory role for PGLYRP1. This is mediated through decreased plasmacytoid dendritic cells (pDC) and regulatory T cells (Treg) in wild type mice .

  • Lung Damage Model: Conversely, PGLYRP1/Tag7 peptides decrease proinflammatory responses in models of diffuse alveolar damage, suggesting potential anti-inflammatory properties .

• Context-Dependent Protein Interaction Analysis:

  • Use PGLYRP1 Antibody, Biotin conjugated to immunoprecipitate PGLYRP1 complexes from different tissue environments

  • Perform mass spectrometry to identify context-specific binding partners

  • Validate interactions through co-immunoprecipitation and proximity ligation assays

• Structure-Function Relationship Studies:

  • Investigate whether full-length PGLYRP1 versus specific peptide fragments have different immunomodulatory effects

  • Create truncation mutants and test their activity in different disease models

  • Assess how post-translational modifications affect PGLYRP1's functional outcomes

• Receptor Competition Experiments:

  • Determine if PGLYRP1 binding to different receptors (TREM-1 vs. TNFR1) results in different downstream effects

  • Use receptor-specific blocking antibodies to assess which signaling pathways predominate in different tissues

Experimental Design Table:

This comprehensive approach would help resolve the apparent contradictions and provide a more nuanced understanding of how PGLYRP1 functions as an immunomodulatory molecule in different pathological contexts.

What are the most common technical challenges when using PGLYRP1 Antibody, Biotin conjugated and how can they be addressed?

Researchers working with PGLYRP1 Antibody, Biotin conjugated may encounter several technical challenges that can affect experimental outcomes. Here are methodological solutions for addressing the most common issues:

1. High Background Signal:

• Cause: Insufficient blocking or non-specific binding of the biotin-conjugated antibody

• Solution:

  • Increase blocking time and concentration (use 5% BSA or normal serum)

  • Include a dedicated biotin blocking step to minimize endogenous biotin interference

  • Optimize antibody dilution (test range from 1:50 to 1:1,000)

  • Increase washing steps (6-8 washes instead of standard 4)

2. Weak or No Signal:

• Cause: Degraded antibody, inappropriate storage, or target protein denaturation

• Solution:

  • Verify antibody activity with a positive control sample

  • Ensure proper storage conditions (-20°C or -80°C, avoiding freeze-thaw cycles)

  • For ELISA, ensure the capture antibody recognizes a different epitope than the biotin-conjugated detection antibody

  • Optimize incubation conditions (temperature and duration)

3. Cross-Reactivity Issues:

• Cause: Antibody binding to proteins with similar epitopes

• Solution:

  • Validate specificity using PGLYRP1 knockout or knockdown samples

  • Pre-absorb antibody with recombinant PGLYRP family members (especially PGLYRP3, an important paralog)

  • Use more stringent washing conditions (higher salt concentration)

4. Biotin-Streptavidin System Complications:

• Cause: Inefficient binding of streptavidin to biotin-conjugated antibody

• Solution:

  • Ensure streptavidin reagent is active and properly stored

  • Optimize streptavidin-HRP concentration and incubation time

  • Consider using different detection systems (e.g., ABC complex) for signal amplification

5. Reproducibility Issues:

• Cause: Variations in experimental conditions or reagent quality

• Solution:

  • Standardize all protocol steps, including incubation times and temperatures

  • Prepare master mixes of reagents for multiple samples

  • Use the same lot of antibody for comparative experiments when possible

Implementing these troubleshooting approaches will help ensure more consistent and reliable results when working with PGLYRP1 Antibody, Biotin conjugated across various experimental applications.

How should researchers validate the specificity of PGLYRP1 Antibody, Biotin conjugated in their experimental systems?

Validating antibody specificity is crucial for ensuring the reliability of experimental results. For PGLYRP1 Antibody, Biotin conjugated, implement the following comprehensive validation strategy:

Methodological Approach to Antibody Validation:

• Genetic Controls:

  • Test antibody on samples from Pglyrp1 knockout models

  • Use PGLYRP1 siRNA knockdown in relevant cell lines

  • Compare staining patterns between known high and low PGLYRP1-expressing tissues

• Peptide Competition Assay:

  • Pre-incubate antibody with excess immunizing peptide (corresponding to residues near the C-terminal of human peptidoglycan recognition protein 1)

  • Compare signal between peptide-blocked and unblocked antibody

  • Significant signal reduction confirms specificity for the target epitope

• Western Blot Validation:

  • Confirm recognition of protein at expected molecular weight (calculated MW: 22)

  • Look for single band rather than multiple non-specific bands

  • Compare pattern with other validated anti-PGLYRP1 antibodies

• Orthogonal Method Validation:

  • Correlate protein detection with mRNA expression (qPCR)

  • Compare results with mass spectrometry data where available

  • Use alternative detection methods like RNA-FISH to confirm expression patterns

• Cross-Reactivity Assessment:

  • Test against recombinant PGLYRP family members (PGLYRP2, PGLYRP3, PGLYRP4)

  • Assess species cross-reactivity if working with non-human models

  • Document any observed cross-reactivity in your experimental system

Validation Protocol Flowchart:

• Initial testing on positive control samples (human neutrophils express high levels of PGLYRP1)

• Parallel testing with genetic knockdown/knockout samples

• Peptide competition assay to confirm epitope specificity

• Cross-reference with orthogonal methods

• Final validation in the specific experimental system/tissue of interest

Following this systematic validation approach will ensure that your experimental findings accurately reflect PGLYRP1 biology rather than artifacts from non-specific antibody binding.

What emerging research areas could benefit from applications of PGLYRP1 Antibody, Biotin conjugated?

PGLYRP1 research intersects with multiple cutting-edge fields in biomedical science. The availability of specific tools like PGLYRP1 Antibody, Biotin conjugated enables several promising research directions:

1. Therapeutic Development for Asthma:
Research has established that PGLYRP1 contributes to asthma pathogenesis, with Pglyrp1-/- mice developing less severe disease than wild type counterparts . This creates opportunities to:

• Screen for small molecule inhibitors of PGLYRP1 using biotin-conjugated antibodies in competitive binding assays

• Develop antibody-based therapeutics that neutralize PGLYRP1 activity

• Investigate how PGLYRP1 blockade affects plasmacytoid dendritic cell (pDC) and regulatory T cell (Treg) populations in asthmatic lungs

2. Cancer Immunotherapy Approaches:
PGLYRP1's role in forming complexes with heat shock protein HSPA1A that induce tumor cell death suggests potential applications in:

• Developing cancer immunotherapies that enhance PGLYRP1-mediated tumor cell killing

• Creating diagnostic tools to assess PGLYRP1 expression in tumor samples as a potential biomarker

• Investigating how PGLYRP1 modulates immune checkpoint pathways in the tumor microenvironment

3. Inflammatory Disease Modulation:
The dual pro-inflammatory and anti-inflammatory effects of PGLYRP1 in different contexts warrant investigation into:

• Structure-function relationships determining these opposing activities

• Development of context-specific PGLYRP1 modulators

• Therapeutic applications in inflammatory bowel disease, psoriasis, and rheumatoid arthritis

4. Antimicrobial Resistance Countermeasures:
PGLYRP1's natural bactericidal activity against Gram-positive bacteria and bacteriostatic effects on Gram-negative bacteria offers potential for:

• Developing novel antimicrobial agents based on PGLYRP1's mechanism of action

• Creating diagnostic tools to monitor PGLYRP1 expression during infection

• Investigating PGLYRP1's role in maintaining microbiome homeostasis

5. Neurodegenerative Disease Research:
The involvement of PGLYRP1 in neuroscience research areas suggests unexplored roles in:

• Neuroinflammatory processes underlying conditions like Alzheimer's and Parkinson's diseases

• Interactions between the nervous system and innate immunity

• Potential diagnostic applications for neuroinflammatory conditions

These emerging research directions highlight the versatility of PGLYRP1 Antibody, Biotin conjugated as a tool for investigating complex biological processes with significant therapeutic implications.

How might advances in single-cell technologies enhance our understanding of PGLYRP1 expression and function?

Single-cell technologies offer unprecedented opportunities to dissect the heterogeneity of PGLYRP1 expression and function across diverse cell populations. Integrating PGLYRP1 Antibody, Biotin conjugated into these advanced platforms could revolutionize our understanding:

Methodological Applications in Single-Cell Research:

• Single-Cell Proteomics:

  • Adapting biotin-conjugated PGLYRP1 antibodies for CyTOF (mass cytometry) to simultaneously detect PGLYRP1 alongside dozens of other proteins

  • Incorporating into CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) to correlate PGLYRP1 protein expression with transcriptome-wide gene expression

  • Developing antibody-based single-cell Western blotting for PGLYRP1 detection in rare cell populations

• Spatial Transcriptomics Integration:

  • Using PGLYRP1 Antibody, Biotin conjugated in multiplexed immunofluorescence to map protein expression in tissue microenvironments

  • Correlating with spatial transcriptomics data to understand how tissue context influences PGLYRP1 expression

  • Investigating cell-cell communication networks involving PGLYRP1-expressing cells

• Functional Single-Cell Assays:

  • Developing microfluidic approaches to assess PGLYRP1 secretion at the single-cell level

  • Measuring the effects of PGLYRP1 on individual target cells using high-content imaging

  • Creating reporter systems to monitor PGLYRP1-mediated signaling events in real-time

Potential Research Questions Addressable Through Single-Cell Approaches:

These approaches would help resolve current controversies regarding PGLYRP1's seemingly contradictory roles in different disease contexts. By identifying specific cell populations in which PGLYRP1 exerts pro-inflammatory versus anti-inflammatory effects, researchers could develop more targeted therapeutic strategies for conditions like asthma and diffuse alveolar damage .