PRKCB Antibody, Biotin conjugated

What is PRKCB and what role does it play in cellular functions?

PRKCB (Protein Kinase C Beta) is a member of the AGC Ser/Thr protein kinase family that plays key roles in numerous cellular processes. It functions as a signaling molecule involved in secretion, gene expression, proliferation, and muscle contraction . In the immune system, PRKCB is critically important for B cell activation, germinal center formation, and plasma cell development . The protein consists of 671-673 amino acid residues with a molecular weight of approximately 77 kDa . PRKCB has multiple subcellular localizations including membrane, nucleus, and cytoplasm, reflecting its diverse functions .

The protein exhibits high expression in lymphoid tissues, particularly the lymph node and spleen, consistent with its prominent role in B cell biology . PRKCB-null (Prkcb−/−) mice are severely immunodeficient, demonstrating the essential nature of this kinase in immune function . At the cellular level, PRKCB regulates antigen polarization, mTORC1 signaling, metabolic reprogramming, and mitochondrial remodeling in B cells, functioning as a master regulator that dictates B cell fate decisions .

What are the different PRKCB isoforms and how do they differ functionally?

PRKCB exists in up to two different isoforms (PRKCB1 and PRKCB2, also known as PKCβI and PKCβII), which arise from alternative splicing of the PRKCB gene . These isoforms differ in their C-terminal regions, resulting in distinct subcellular localizations and functions. The PRKCB protein belongs to the classical PKC subfamily that requires calcium, diacylglycerol, and phospholipids for activation .

Functionally, PRKCB isoforms have been reported to regulate various cellular processes including:

• B cell activation and differentiation

• Apoptosis induction

• Endothelial cell proliferation

• Intestinal sugar absorption

• Neuronal functions related to fear-induced conflict behavior after stress

When designing experiments with PRKCB antibodies, researchers should consider which isoform(s) their antibody recognizes and how this might impact data interpretation, especially when studying tissues where both isoforms may be differentially expressed.

Why should I choose a biotin-conjugated PRKCB antibody for my research?

Biotin-conjugated PRKCB antibodies offer several advantages for research applications:

• Signal amplification: The strong interaction between biotin and streptavidin/avidin provides significant signal enhancement, which is particularly valuable when detecting low-abundance PRKCB expression.

• Flexibility in detection systems: Biotin-conjugated antibodies can be detected using various streptavidin-conjugated reporters (fluorescent dyes, enzymes, quantum dots), allowing for versatility in experimental design.

• Multicolor detection: In co-localization studies, biotin-conjugated PRKCB antibodies can be paired with directly labeled antibodies against other targets, enabling complex multi-parameter analyses.

• Cost-effectiveness: A single biotin-conjugated primary antibody can be used with different detection systems without requiring multiple directly-conjugated antibodies.

Commercial suppliers offer biotin-conjugated PRKCB antibodies that have been validated for multiple applications including Western blot, flow cytometry, and immunohistochemistry .

What are the optimal sample preparation methods for detecting PRKCB in lymphoid tissues?

For optimal detection of PRKCB in lymphoid tissues such as spleen and lymph nodes, consider the following methodological approaches:

For frozen sections:

• Harvest tissue and immediately snap-freeze in OCT compound using liquid nitrogen-cooled isopentane.

• Cut 5-8 μm sections and fix with 4% paraformaldehyde for 10 minutes at room temperature.

• Include a permeabilization step (0.1-0.5% Triton X-100) to improve antibody access to intracellular PRKCB.

• Block endogenous biotin using a commercial biotin blocking kit to minimize background.

• Incubate with biotin-conjugated PRKCB antibody overnight at 4°C.

• Detect using fluorophore-conjugated streptavidin.

For FFPE (formalin-fixed paraffin-embedded) tissues:

• Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0).

• Block endogenous peroxidase activity with 3% H₂O₂ if using enzymatic detection methods.

• Include a streptavidin/biotin blocking step to minimize background.

• Incubate with biotin-conjugated PRKCB antibody (diluted 1:200-1:500).

• Detect using HRP-streptavidin and DAB substrate.

When analyzing B cells specifically, consider co-staining with B cell markers like B220 to properly identify the population of interest, as demonstrated in studies of germinal center formation in PRKCB-deficient mice .

How should I optimize Western blot protocols for PRKCB detection?

For successful detection of PRKCB by Western blot, follow these methodological recommendations:

• Sample preparation:

  • Lyse cells in RIPA buffer supplemented with protease and phosphatase inhibitors

  • Include 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 mM sodium orthovanadate

  • Sonicate briefly and centrifuge at 14,000 × g for 15 minutes at 4°C

• Gel electrophoresis and transfer:

  • Load 20-40 μg of protein per lane on 8-10% SDS-PAGE gels

  • Transfer to PVDF membranes (preferred over nitrocellulose for PRKCB detection)

  • Use wet transfer systems at 100V for 90 minutes for optimal transfer of this 77 kDa protein

• Antibody incubation:

  • Block membranes with 5% non-fat dry milk in TBST for 1 hour

  • Incubate with biotin-conjugated PRKCB antibody (1:1000 dilution) overnight at 4°C

  • Wash extensively with TBST (3 × 10 minutes)

  • Incubate with HRP-conjugated streptavidin (1:5000) for 1 hour at room temperature

• Detection:

  • Develop using enhanced chemiluminescence reagents

  • Expected band size: approximately 77 kDa

When analyzing phosphorylated forms of PRKCB, consider using a dual detection approach with antibodies specific to phosphorylated and total PRKCB to accurately assess activation status.

What controls should I include when using biotin-conjugated PRKCB antibodies?

Including appropriate controls is crucial for reliable interpretation of experiments using biotin-conjugated PRKCB antibodies:

Essential controls:

• Positive control: Samples known to express PRKCB, such as lymph node or spleen tissue sections, or B cell lines like Raji cells.

• Negative control: Samples with minimal PRKCB expression or PRKCB knockout tissues/cells. Studies with Prkcb−/− mice provide excellent negative controls for antibody validation .

• Isotype control: Include a biotin-conjugated isotype-matched control antibody (e.g., mouse IgG1 for mouse monoclonal antibodies) to assess non-specific binding .

• Secondary-only control: Omit the primary antibody but include the streptavidin detection reagent to evaluate background from the detection system.

• Biotin blocking control: For tissues with high endogenous biotin (e.g., liver, kidney), compare results with and without a biotin blocking step to assess endogenous biotin contribution to signal.

Additional validation approaches:

• Peptide competition assays to confirm antibody specificity

• Comparison of results across multiple PRKCB antibodies targeting different epitopes

• Correlation of protein detection with mRNA expression data

Including these controls enhances data reliability and facilitates troubleshooting if unexpected results occur.

How can I use PRKCB antibodies to investigate B cell fate decisions in germinal centers?

PRKCB plays a critical role in B cell fate decisions, particularly in germinal center formation and plasma cell differentiation. To investigate these processes:

• Analysis of germinal center formation:

  • Immunize mice with T-dependent antigens like NP-KLH in alum

  • Collect spleens at day 13 post-immunization

  • Prepare tissue sections and stain for:

    • PRKCB (biotin-conjugated antibody)

    • GL7 and CD95 (germinal center markers)

    • B220 (B cell marker)

  • Quantify both the number and area of germinal centers

• B cell fate marker analysis by flow cytometry:

  • Isolate B cells from spleen or lymph nodes

  • Stimulate with appropriate conditions (e.g., anti-IgM, CD40L, and IL-4)

  • Analyze expression of:

    • PRKCB (intracellular staining)

    • PAX5 (B cell identity factor)

    • IRF4 (plasma cell differentiation factor)

    • Other relevant markers (BLIMP1, XBP1)

  • Track the IRF4hiPAX5lo plasma cell signature over time

• Analysis of metabolic reprogramming:

  • PRKCB regulates mitochondrial remodeling and heme biosynthesis during B cell activation

  • Measure mitochondrial mass using MitoTracker dyes

  • Assess oxygen consumption rate and extracellular acidification rate

  • Correlate PRKCB expression with metabolic parameters to understand how this kinase regulates B cell fate through metabolic control

This multi-parameter approach allows comprehensive assessment of how PRKCB influences the germinal center reaction and plasma cell differentiation, providing insights into B cell immunology.

What are the best approaches for studying PRKCB-mediated antigen polarization in B cells?

PRKCB plays a crucial role in antigen polarization within B cells, a process important for efficient antigen presentation to T cells. To investigate this phenomenon:

• Visualization of antigen polarization:

  • Isolate primary B cells from spleen or lymph nodes

  • Stimulate with fluorescently-labeled anti-IgM (e.g., Alexa647-conjugated) for 30 minutes

  • Fix cells with 4% paraformaldehyde

  • Counterstain with DAPI and phalloidin to visualize nuclei and actin cytoskeleton

  • Analyze using confocal microscopy, quantifying the percentage of cells with polarized antigen compartments

• Antigen presentation assays:

  • Stimulate B cells with antigen-coated microspheres (e.g., Eα peptide)

  • Detect presentation using anti-MHC-II:peptide complex antibodies

  • Measure by flow cytometry, comparing mean fluorescence intensity between wild-type and PRKCB-deficient B cells

  • Alternatively, use OT-II T cell proliferation assays to measure functional consequences of altered antigen polarization

• Synergistic BCR and TLR9 signaling:

  • Culture B cells with anti-IgM and CpG-coated microspheres

  • Assess proliferation using CellTrace Violet dilution

  • Compare responses between wild-type and PRKCB-deficient B cells

  • Include controls with unlinked CpG and anti-IgM to distinguish polarization-dependent effects

This comprehensive approach allows detailed characterization of how PRKCB regulates antigen polarization and its functional consequences for B cell activation and antigen presentation.

How can I detect different phosphorylation states of PRKCB and what is their functional significance?

PRKCB undergoes multiple phosphorylation events that regulate its activation and function. To study these modifications:

• Western blot analysis of phosphorylation sites:

  • Use phospho-specific antibodies targeting key residues:

    • Thr500 (activation loop)

    • Thr641 (turn motif)

    • Ser660 (hydrophobic motif)

  • Compare total PRKCB levels using pan-PRKCB antibodies

  • Evaluate changes in phosphorylation status following B cell activation with anti-IgM, CD40L, or other stimuli

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation

• Immunofluorescence analysis of phospho-PRKCB localization:

  • Use biotin-conjugated phospho-specific PRKCB antibodies

  • Examine subcellular localization changes upon B cell activation

  • Co-stain with markers for specific cellular compartments (plasma membrane, cytoskeleton, mitochondria)

  • Quantify translocation using image analysis software

• Functional correlation:

  • Correlate phosphorylation patterns with:

    • mTORC1 activation (phospho-S6K, phospho-4E-BP1)

    • Antigen polarization

    • Mitochondrial remodeling

    • Germinal center formation and plasma cell differentiation

• Phosphomimetic and phosphodeficient mutants:

  • Introduce PRKCB constructs with mutations at key phosphorylation sites into PRKCB-deficient B cells

  • Assess rescue of phenotypes including antigen polarization and plasma cell differentiation

  • Determine which phosphorylation events are critical for specific PRKCB functions

This methodological approach provides a comprehensive understanding of how phosphorylation regulates PRKCB activity and its role in B cell biology.

Why might I observe high background when using biotin-conjugated PRKCB antibodies in certain tissues?

High background is a common challenge when using biotin-conjugated antibodies. Several factors may contribute to this issue:

• Endogenous biotin in tissues:

  • Many tissues (especially liver, kidney, brain) contain endogenous biotin

  • Solution: Implement a biotin blocking step using commercial biotin blocking kits before applying the biotin-conjugated PRKCB antibody

  • Alternative approach: Use avidin-biotin blocking system with sequential application of avidin and biotin

• Endogenous biotin-binding proteins:

  • Tissues may contain proteins that naturally bind biotin or streptavidin

  • Solution: Include a pre-incubation with unconjugated streptavidin followed by free biotin to block these interactions

• Fc receptor binding:

  • B cells express Fc receptors that can bind antibodies non-specifically

  • Solution: Include Fc receptor blocking reagents (e.g., normal serum from the same species as the secondary reagent, or commercial Fc block)

• Suboptimal blocking:

  • Insufficient blocking can lead to non-specific binding

  • Solution: Optimize blocking conditions using various reagents (BSA, normal serum, commercial blocking solutions) and longer incubation times

• Cross-reactivity:

  • The PRKCB antibody may cross-react with other PKC isoforms

  • Solution: Validate antibody specificity using PRKCB knockout tissues/cells as negative controls

Implementing these methodological refinements can significantly improve signal-to-noise ratio when working with biotin-conjugated PRKCB antibodies.

What should I do if my PRKCB antibody does not detect the expected 77 kDa band in Western blot?

If you fail to detect the expected 77 kDa PRKCB band in Western blot, consider these methodological troubleshooting approaches:

• Protein extraction method:

  • Certain lysis buffers may not efficiently extract membrane-associated PRKCB

  • Try RIPA buffer with 0.1% SDS or specialized membrane protein extraction kits

  • Ensure samples are thoroughly sonicated to disrupt membrane structures

• Protein degradation:

  • PRKCB may be degraded during sample preparation

  • Add a comprehensive protease inhibitor cocktail to lysis buffers

  • Keep samples cold throughout processing

  • Avoid repeated freeze-thaw cycles

• Transfer efficiency:

  • Large proteins like PRKCB (77 kDa) may transfer inefficiently

  • Use lower percentage gels (8%)

  • Extend transfer time or use specialized transfer conditions for larger proteins

  • Consider semi-dry vs. wet transfer optimization

• Antibody specificity:

  • Verify antibody specificity using positive control lysates (lymph node tissue, B cell lines)

  • Compare results with alternative PRKCB antibodies targeting different epitopes

  • Check if the antibody recognizes specific PRKCB isoforms (PRKCB1 vs. PRKCB2)

• Protein expression levels:

  • PRKCB expression might be below detection threshold

  • Increase protein loading (50-80 μg)

  • Use enhanced chemiluminescence substrates with higher sensitivity

  • Consider immunoprecipitation to enrich PRKCB before Western blot analysis

Systematic evaluation of these factors will help resolve Western blot detection issues for PRKCB.

How can I reconcile conflicting data between PRKCB protein expression and functional outcomes?

Researchers sometimes encounter discrepancies between PRKCB protein expression levels and observed functional outcomes. To address such conflicts:

• Isoform-specific effects:

  • Different PRKCB isoforms (PRKCB1/PRKCB2) may have distinct functions

  • Use isoform-specific antibodies to distinguish between PRKCB variants

  • Correlate specific isoform expression with functional outcomes

• Activation state vs. expression level:

  • PRKCB activity depends on phosphorylation state, not just expression level

  • Measure phosphorylation at key regulatory sites (Thr500, Thr641, Ser660)

  • Assess membrane translocation as an indicator of activation

  • Use kinase activity assays to directly measure PRKCB enzymatic activity

• Compensatory mechanisms:

  • Other PKC isoforms may compensate for PRKCB deficiency

  • Examine expression of related family members (especially PKCα)

  • Compare phenotypes between single (PRKCB) and double (PRKCB + other PKC) knockout models

• Context-dependent functions:

  • PRKCB functions may vary depending on cell type and activation state

  • Study PRKCB in defined B cell subpopulations (naive, germinal center, plasma cells)

  • Compare PRKCB function across different stimulation conditions

  • Consider the influence of microenvironment on PRKCB function

• Technical considerations:

  • Antibody specificity and sensitivity can affect detection

  • Validate key findings using complementary techniques (protein, mRNA, functional assays)

  • Consider spatial and temporal aspects of PRKCB expression and activation

How can PRKCB antibodies be used to investigate the role of this kinase in metabolic reprogramming of B cells?

Recent research has revealed that PRKCB regulates metabolic reprogramming in B cells, particularly during plasma cell differentiation. To investigate this emerging area:

• Mitochondrial analyses:

  • Use biotin-conjugated PRKCB antibodies for co-localization studies with mitochondrial markers

  • Assess changes in PRKCB-mitochondria association during B cell activation

  • Measure mitochondrial parameters (mass, membrane potential) in relation to PRKCB expression

  • Analyze mitochondrial remodeling during B cell differentiation

• mTORC1 signaling assessment:

  • PRKCB regulates mTORC1 activity, which controls metabolic reprogramming

  • Measure phosphorylation of mTORC1 targets (S6K, 4E-BP1) in relation to PRKCB activation

  • Use flow cytometry or Western blot to correlate PRKCB levels with mTORC1 activity

  • Determine how biotin-conjugated PRKCB antibodies can track changes in PRKCB localization during mTORC1 activation

• Heme biosynthesis pathway:

  • PRKCB promotes heme biosynthesis during plasma cell differentiation

  • Investigate PRKCB regulation of key enzymes in this pathway

  • Correlate PRKCB expression with heme levels during B cell differentiation

  • Study how PRKCB links metabolic changes to BLIMP1-driven plasma cell development

This approach provides comprehensive insights into how PRKCB orchestrates metabolic changes required for B cell differentiation and antibody production.

What are the most effective multiplex immunofluorescence approaches for studying PRKCB in lymphoid tissues?

Multiplex immunofluorescence allows simultaneous detection of multiple markers alongside PRKCB, providing rich contextual information:

• Sequential multiplex immunofluorescence:

  • Use biotin-conjugated PRKCB antibody with fluorescent streptavidin detection

  • Combine with directly conjugated antibodies against B cell markers (CD19, B220)

  • Add germinal center markers (GL7, CD95) and plasma cell markers (CD138)

  • Implement tyramide signal amplification for enhanced sensitivity

  • Include markers for T cells (CD3, CD4) to analyze B-T cell interactions

• Antibody stripping and reprobing protocols:

  • Perform initial staining with biotin-PRKCB and fluorescent detection

  • Image the section

  • Strip antibodies using optimized buffer (glycine-SDS, pH 2.0)

  • Reprobe with additional antibodies

  • Register and overlay multiple rounds of imaging

• Spectral unmixing approaches:

  • Use biotin-PRKCB with streptavidin-conjugated spectrally distinct fluorophores

  • Combine with multiple directly-labeled antibodies

  • Acquire data using spectral detectors

  • Apply computational unmixing algorithms to separate overlapping fluorophores

  • Analyze co-expression patterns using multi-parameter analysis tools

This multiplex approach allows comprehensive characterization of PRKCB expression in complex lymphoid tissues and provides insight into its role in immune responses.