PRKCB Antibody, HRP conjugated

What is PRKCB and what cellular functions does it regulate?

PRKCB (Protein Kinase C Beta) is a serine/threonine-protein kinase of the protein kinase C family that plays crucial roles in multiple cellular processes. It functions as a key regulator of B cell polarity and activation and influences autophagy levels . PRKCB has been implicated in the pathogenesis of several diseases, including nontuberculous mycobacterial pulmonary disease (NTM-PD) . At the molecular level, PRKCB can inhibit the fusion between lysosomes and mycobacterial phagosomes, thereby promoting the intracellular survival of pathogens such as M. avium . This protein interacts with multiple components involved in vesicle fusion and protein localization to vacuoles or lysosomes, including vacuolar protein sorting-associated protein 16 homolog (VPS16) and the G subunit of V-ATPase (ATP6V1G) .

What are the optimal storage conditions for PRKCB antibody, HRP conjugated?

For maximum stability and reactivity of PRKCB antibody (HRP conjugated), store the product at -20°C or -80°C upon receipt . It is critical to avoid repeated freeze-thaw cycles as these can significantly compromise antibody functionality . The antibody is provided in a liquid form with a buffer containing preservative (0.03% Proclin 300) and constituents (50% Glycerol, 0.01M PBS, pH 7.4) that help maintain stability . For laboratory protocols requiring regular use, consider preparing working aliquots to minimize exposure to damaging conditions. The stability of the antibody is determined by the rate of activity loss, which should be less than 5% within the expiration date under appropriate storage conditions .

What applications is the PRKCB antibody, HRP conjugated most suitable for?

The PRKCB antibody, HRP conjugated, is primarily validated for Enzyme-Linked Immunosorbent Assay (ELISA) applications . The antibody demonstrates specific reactivity to human PRKCB, making it particularly valuable for studies focusing on human samples or cell lines . While the primary validated application is ELISA, researchers may optimize conditions for other applications such as Western blotting, immunoprecipitation, or immunohistochemistry, though additional validation would be necessary. The antibody's high purification level (>95%, Protein G purified) ensures minimal non-specific binding, which is crucial for obtaining reliable and reproducible results in sensitive detection methods .

How should I design experiments to investigate PRKCB's role in phagosome-lysosome fusion?

Based on recent research findings, investigating PRKCB's role in phagosome-lysosome fusion requires a multifaceted experimental approach:

• Co-immunoprecipitation assays: Design experiments to examine the interaction between PRKCB and key components of the phagosome-lysosome fusion machinery, particularly VPS16 and ATP6V1G1 . These assays can be coupled with Western blotting using the HRP-conjugated PRKCB antibody for detection.

• Confocal microscopy: Implement immunofluorescence studies to visualize colocalization of PRKCB with VPS16 and ATP6V1G in cells exposed to pathogens like M. avium . This requires careful optimization of fixation and permeabilization protocols to preserve protein interactions.

• Phagosomal acidification assays: Measure the impact of PRKCB expression/inhibition on phagosomal pH using LysoTracker or pH-sensitive fluorescent proteins in macrophage models .

• Infection models: Establish M. avium infection protocols in macrophages with varying levels of PRKCB expression to quantify intracellular bacterial survival rates .

• Protein-protein interaction validation: Confirm the functional significance of identified interactions through mutational analysis of key domains in PRKCB that mediate its binding to VPS16 and ATP6V1G .

Include appropriate controls such as isotype-matched antibodies and PRKCB knockdown/knockout cells to ensure specificity and validity of observations.

What are the optimal dilutions and sample preparation methods for detecting PRKCB using HRP-conjugated antibodies?

While optimal dilutions should be determined by the end user through titration experiments, the following guidelines can help establish a starting point:

For ELISA applications:

• Primary antibody dilution: Start with 1:500 to 1:2000 range

• Sample preparation varies by sample type:

  Sample Type	Preparation Method	Recommended Dilution
  Serum	Centrifuge at 1000×g for 10 min	1:2 to 1:10
  Plasma	Use EDTA/heparin as anticoagulant, centrifuge at 1000×g for 15 min	1:2 to 1:10
  Cell lysates	Sonicate or freeze-thaw cycles followed by centrifugation at 10,000×g for 10 min	Depends on expression level
  Tissue homogenates	Homogenize in PBS (pH 7.4) and centrifuge at 10,000×g for 5 min	1:2 to 1:20

For quantitative detection of PRKCB, sandwich ELISA technologies demonstrate high sensitivity, with detection limits below 0.07 ng/ml and a testing range of 0.156 ng/ml - 10 ng/ml . To minimize background and enhance specificity, ensure blocking buffers contain 1-5% BSA or normal serum from the same species as the secondary antibody, and include appropriate washing steps between incubations .

How can I resolve issues with high background when using PRKCB antibody, HRP conjugated?

High background is a common issue when working with HRP-conjugated antibodies. A systematic approach to troubleshooting includes:

• Optimize antibody concentration: Excessive antibody concentration is a leading cause of background signal. Perform a titration experiment using dilutions ranging from 1:250 to 1:5000 to determine the optimal signal-to-noise ratio .

• Improve blocking conditions: Increase blocking reagent concentration (3-5% BSA or normal serum) and extend blocking time to 1-2 hours at room temperature. Ensure the blocking agent is from a species different from that of the primary antibody .

• Adjust washing protocol: Implement more stringent washing steps between incubations using TBS-T (0.05-0.1% Tween-20). Increase both the number of washes (5-6 times) and duration (5 minutes each) .

• Reduce incubation temperature: Consider incubating at 4°C overnight rather than at room temperature to enhance specificity of binding.

• Use additives to reduce non-specific binding: Add 0.1-0.5% Tween-20 or 0.1-1% Triton X-100 to antibody dilution buffers to reduce hydrophobic interactions.

• Optimize substrate development: For HRP-based detection systems, reduce substrate incubation time and protect from light during development to minimize excessive signal generation .

If background persists despite these optimizations, consider additional purification of samples or alternative detection methods.

What are the critical controls needed when studying PRKCB's interaction with VPS16 and ATP6V1G?

When investigating PRKCB's interactions with VPS16 and ATP6V1G in the context of phagosome-lysosome fusion, several critical controls should be implemented:

• Negative interaction controls:

  • IgG isotype control immunoprecipitations to identify non-specific binding

  • Immunoprecipitation using lysates from PRKCB knockout/knockdown cells

  • Competitive blocking with recombinant PRKCB protein

• Positive interaction controls:

  • Co-immunoprecipitation of known PRKCB interacting partners

  • Reciprocal immunoprecipitations (pull down with anti-VPS16 or anti-ATP6V1G)

• Functional validation controls:

  • PRKCB inhibitor treatments to confirm kinase activity dependence

  • Phosphorylation-dead mutants of PRKCB to determine if kinase activity is required

  • Domain deletion mutants to map interaction regions

• Specificity controls:

  • Testing interactions with other PKC family members to confirm specificity

  • Analysis of interactions in different cell types and conditions

  • Domain-specific antibodies to confirm interaction regions

• Biological relevance controls:

  • Parallel analysis of phagosome acidification

  • Bacterial survival assays with and without PRKCB inhibition

  • Colocalization analysis during active infection versus non-infected conditions

Implementation of these controls ensures that observed interactions are specific, reproducible, and biologically relevant to the phagosome-lysosome fusion process.

How can PRKCB antibody be used to investigate its role in pathogen survival mechanisms?

PRKCB antibody can be strategically deployed to elucidate its role in pathogen survival through several advanced experimental approaches:

• Temporal dynamics of PRKCB recruitment: Use the HRP-conjugated PRKCB antibody in time-course experiments to monitor PRKCB recruitment to phagosomes containing pathogens like M. avium. This can be accomplished using cell fractionation followed by Western blotting or high-resolution microscopy with appropriate markers for different phagosomal maturation stages .

• Phosphoproteomic analysis: Employ the antibody for immunoprecipitation of PRKCB from infected versus uninfected cells, followed by mass spectrometry to identify differentially phosphorylated substrates that may be involved in phagosome maturation arrest .

• ChIP-seq applications: Use the antibody in chromatin immunoprecipitation followed by sequencing to identify transcriptional targets regulated by nuclear PRKCB during infection, revealing potential host-response mechanisms.

• Proximity labeling techniques: Combine the antibody with proximity labeling approaches (BioID or APEX) to identify the complete interactome of PRKCB during infection, potentially revealing novel mediators of pathogen survival.

• Single-cell analysis: Apply the antibody in flow cytometry or mass cytometry approaches to correlate PRKCB expression/activation levels with pathogen burden at the single-cell level .

These approaches can reveal how pathogens might exploit PRKCB to enhance their survival within host cells and identify potential intervention points for therapeutic development.

What methodologies can be used to study PRKCB's impact on B cell fate determination?

Based on recent findings regarding PRKCB's role in B cell fate determination, several methodologies can be employed:

• B cell isolation and culture systems: Establish primary B cell cultures from different developmental stages to study how PRKCB expression/activation affects differentiation trajectories. The HRP-conjugated PRKCB antibody can be used to quantify expression levels in different B cell subsets through Western blotting or intracellular FACS .

• Mitochondrial function assessment: As PRKCB appears to regulate mitochondrial processes in B cells, implement assays measuring:

  • Mitochondrial membrane potential (using JC-1 or TMRE dyes)

  • Oxygen consumption rate (using Seahorse XF analyzer)

  • Mitochondrial ROS production (using MitoSOX)

  • Mitochondrial mass (using MitoTracker dyes)

• Genetic manipulation approaches:

  • CRISPR/Cas9-mediated knockout or knockin of PRKCB in B cell lines

  • Conditional knockout models in mice to study B cell development in vivo

  • Expression of constitutively active or dominant-negative PRKCB mutants

• Signaling pathway analysis:

  • Phospho-flow cytometry to measure activation of downstream targets

  • Quantitative immunoblotting using the HRP-conjugated antibody to assess signaling kinetics

  • RNA-seq to identify transcriptional changes dependent on PRKCB activity

• In vivo B cell fate tracking:

  • Adoptive transfer of PRKCB-modified B cells labeled with cell tracking dyes

  • Lineage tracing using reporter systems in conditional PRKCB knockout mice

These methodologies would provide comprehensive insights into how PRKCB influences B cell fate decisions and mitochondrial regulation in both physiological and pathological contexts .

How can PRKCB antibody be utilized in translational research for nontuberculous mycobacterial pulmonary disease (NTM-PD)?

The PRKCB antibody, HRP conjugated, can be instrumentally applied in translational research for NTM-PD through several research strategies:

• Biomarker development: Utilize the antibody in ELISA-based assays to quantify PRKCB levels in patient samples (serum, bronchoalveolar lavage fluid, or peripheral blood mononuclear cells) and correlate with disease severity, treatment response, or genetic risk factors (such as the rs194800 polymorphism) . This approach may help stratify patients and predict treatment outcomes.

• Therapeutic target validation:

  • Screen PRKCB inhibitors for their ability to restore phagolysosomal fusion in infected macrophages

  • Develop cell-based assays using the antibody to monitor PRKCB activation status in response to potential therapeutics

  • Establish humanized mouse models of NTM-PD to test PRKCB-targeted interventions

• Pharmacodynamic monitoring: Design sandwich ELISA assays using the HRP-conjugated PRKCB antibody to monitor drug efficacy in clinical trial samples by measuring changes in PRKCB expression or phosphorylation status after treatment.

• Genetic association validation: For patients carrying the risk allele of rs194800 that promotes PRKCB expression, use the antibody to confirm increased protein levels in relevant cell types and correlate with cellular phenotypes like impaired phagosome-lysosome fusion .

• Host-pathogen interaction studies: Develop co-culture systems of human macrophages with M. avium where PRKCB-dependent interactions with VPS16 and ATP6V1G can be monitored using the antibody in combination with proximity ligation assays or FRET-based approaches .

These applications bridge fundamental research findings with clinical applications, potentially leading to novel diagnostic and therapeutic approaches for NTM-PD.

What experimental approaches can reveal the mechanism by which PRKCB blocks phagosome-lysosome fusion?

To elucidate the precise mechanism by which PRKCB blocks phagosome-lysosome fusion, researchers can implement the following experimental approaches:

• Structural biology studies:

  • Use purified proteins to determine the crystal structure of PRKCB in complex with VPS16 and/or ATP6V1G

  • Implement hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Perform in silico molecular docking to predict binding sites for targeted mutagenesis

• Kinase activity dependency analysis:

  • Test whether PRKCB's effect on phagosome-lysosome fusion depends on its kinase activity using kinase-dead mutants

  • Identify potential phosphorylation targets on VPS16 or ATP6V1G using phospho-specific antibodies or mass spectrometry

  • Use phosphomimetic and phospho-deficient mutants to confirm functional significance of identified phosphorylation sites

• Live-cell imaging approaches:

  • Implement real-time confocal microscopy with fluorescently tagged PRKCB, VPS16, and ATP6V1G to visualize dynamic interactions during phagosome maturation

  • Use FRET sensors to measure protein-protein interactions in live cells during infection

  • Apply super-resolution microscopy (STORM/PALM) to map nanoscale organization of these proteins at the phagosome membrane

• Reconstitution systems:

  • Develop in vitro membrane fusion assays with purified components to directly test PRKCB's inhibitory effects

  • Use liposome-based systems incorporating recombinant proteins to recapitulate fusion events

• Comparative studies across mycobacterial species:

  • Examine whether PRKCB's inhibitory effect on phagosome-lysosome fusion occurs with other mycobacterial species beyond M. avium

  • Identify potential bacterial factors that might modulate PRKCB activity using bacterial mutant libraries

These approaches would provide mechanistic insights at molecular resolution, potentially revealing specific intervention points for restoring phagosome-lysosome fusion in infected cells.

How should researchers interpret quantitative PRKCB expression data across different cell types and disease states?

When analyzing quantitative PRKCB expression data across different experimental contexts, consider these interpretation frameworks:

• Cell-type specific baselines: Establish normal PRKCB expression ranges for each cell type under study. Expression levels should be normalized to appropriate housekeeping genes or total protein content, with consideration that:

  • Immune cells (particularly B cells) typically express higher PRKCB levels

  • Expression patterns may vary significantly between primary cells and cell lines

  • Subcellular distribution of PRKCB may be as important as total expression levels

• Disease context analysis:

  • In NTM-PD, increased PRKCB expression in monocytes/macrophages correlates with impaired mycobacterial killing

  • Genetic factors like the rs194800 C allele predispose to higher PRKCB expression

  • Changes in PRKCB expression should be interpreted alongside functional readouts (e.g., phagosomal acidification, bacterial killing capacity)

• Statistical considerations:

  • Implement appropriate statistical tests based on data distribution

  • Use large enough sample sizes to account for biological variability

  • Consider multiple testing corrections when analyzing expression across numerous cell types

  • Report effect sizes alongside statistical significance to assess biological relevance

• Integration with other datasets:

  • Correlate protein expression with transcriptomic data to identify potential post-transcriptional regulation

  • Integrate phosphoproteomic data to distinguish between changes in expression versus activation

  • Consider genetic background (particularly rs194800 genotype) when interpreting expression differences between individuals

This multifaceted interpretation approach ensures that PRKCB expression data contributes meaningfully to understanding its role in health and disease.

What statistical approaches are most appropriate for analyzing PRKCB interaction data from co-immunoprecipitation experiments?

When analyzing protein-protein interaction data from co-immunoprecipitation experiments involving PRKCB, VPS16, and ATP6V1G, several statistical approaches should be considered:

• Quantification methods:

  • Densitometric analysis of Western blot bands with normalization to input controls

  • Ratiometric comparisons between experimental conditions and controls

  • Multi-factor normalization accounting for antibody efficiency and protein abundance

• Statistical tests for paired comparisons:

  • Paired t-tests for comparing interactions under two conditions (e.g., infected vs. uninfected)

  • Repeated measures ANOVA for time-course experiments examining interaction dynamics

  • Non-parametric alternatives (Wilcoxon signed-rank test) when normality assumptions are violated

• Correlation analysis:

  • Pearson or Spearman correlation to assess relationships between interaction strength and functional outcomes (e.g., phagosomal acidification, bacterial survival)

  • Multivariate regression to model how multiple factors affect interaction dynamics

• Reproducibility metrics:

  • Coefficient of variation (CV) calculation across technical and biological replicates

  • Intraclass correlation coefficient (ICC) to assess consistency across experiments

  • Bootstrapping approaches to generate confidence intervals for interaction measures

• Advanced analytical frameworks:

  • Bayesian statistical approaches for handling complex interaction networks

  • Machine learning classification to identify patterns in interaction data across experimental conditions

  • Network analysis tools to visualize and quantify changes in the PRKCB interactome

When reporting results, include both raw data and normalized values, clearly state the normalization method, and provide detailed statistical test parameters including sample sizes, degrees of freedom, test statistics, exact p-values, and measures of effect size.

What are the emerging research areas for PRKCB antibody applications in infectious disease research?

Several cutting-edge research directions are emerging for PRKCB antibody applications in infectious disease studies:

• Single-cell proteomics: Applying the PRKCB antibody in mass cytometry (CyTOF) or single-cell Western blotting to identify heterogeneity in PRKCB expression and activation across immune cell populations during infection. This approach could reveal specialized cell subsets that are particularly susceptible to PRKCB-mediated phagosome maturation arrest .

• Spatial proteomics: Implementing multiplexed immunofluorescence or imaging mass cytometry with the PRKCB antibody to map its distribution within infected tissues and its colocalization with pathogen reservoirs. This could identify microanatomical niches where PRKCB-mediated pathogen persistence occurs.

• Extracellular vesicle (EV) analysis: Investigating whether PRKCB is packaged into EVs during infection and how this might influence intercellular communication and disease progression using the antibody for EV characterization.

• Combinatorial therapeutic targeting: Utilizing the antibody to monitor changes in PRKCB expression/activation when testing combination therapies that target both the pathogen and host pathways, potentially overcoming antimicrobial resistance.

• Cross-pathogen comparative analyses: Extending PRKCB research beyond mycobacteria to other intracellular pathogens that may exploit similar phagosome maturation arrest mechanisms, using the antibody to identify common and distinct regulatory mechanisms .

• Microbiome interactions: Exploring how commensal microbiota influence PRKCB expression in mucosal immune cells and how this affects susceptibility to pathogenic infection, potentially identifying microbiome-based interventions.

These emerging areas represent the frontier of PRKCB research in infectious diseases, with the HRP-conjugated antibody serving as a valuable tool for these investigations.

How might PRKCB inhibitors be developed and tested as potential host-directed therapies for mycobacterial infections?

The development of PRKCB inhibitors as host-directed therapies for mycobacterial infections involves a systematic research pipeline:

• Inhibitor discovery and optimization:

  • High-throughput screening of compound libraries against PRKCB kinase activity

  • Structure-guided design based on PRKCB crystal structures

  • Medicinal chemistry optimization for potency, selectivity, and pharmacokinetic properties

  • Initial testing using the HRP-conjugated PRKCB antibody to confirm target engagement

• In vitro efficacy assessment:

  • Validation in macrophage infection models with diverse mycobacterial species

  • Functional readouts including:

    • Phagosome-lysosome fusion (measured by colocalization assays)

    • Phagosomal acidification (using pH-sensitive dyes)

    • Bacterial survival and replication rates

    • Host cell viability and inflammatory responses

• Mechanism of action studies:

  • Confirming that inhibitors restore VPS16-ATP6V1G interaction using co-immunoprecipitation

  • Phosphoproteomic analysis to identify inhibitor effects on PRKCB substrate phosphorylation

  • Transcriptomic profiling to assess broader impact on host defense pathways

• In vivo efficacy evaluation:

  • Pharmacokinetic/pharmacodynamic (PK/PD) studies to establish dosing regimens

  • Efficacy testing in animal models of mycobacterial infection

  • Combination studies with conventional antimycobacterial agents

  • Assessment of resistance development and long-term efficacy

• Translational and clinical development:

  • Biomarker development using the PRKCB antibody to identify patients most likely to benefit

  • Safety assessment focusing on potential immunological side effects

  • Clinical trial design prioritizing patients with PRKCB-associated genetic risk factors (rs194800 C allele)

  • Development of companion diagnostics to measure PRKCB activity

This systematic approach creates a path from fundamental discoveries about PRKCB's role in mycobacterial persistence to clinically viable host-directed therapies that could address challenges like antimicrobial resistance.