PRKCB Antibody, FITC conjugated

What is PRKCB and why is it significant in cellular research?

PRKCB (Protein Kinase C Beta) is a calcium-activated, phospholipid- and diacylglycerol (DAG)-dependent serine/threonine-protein kinase encoded by the PRKCB gene. It plays crucial roles in multiple cellular processes including regulation of the B-cell receptor (BCR) signalosome, oxidative stress-induced apoptosis, androgen receptor-dependent transcription regulation, insulin signaling, and endothelial cell proliferation . The significance of PRKCB lies in its central position in signal transduction pathways, particularly its key role in B-cell activation through regulating BCR-induced NF-kappa-B activation. It mediates the activation of the canonical NF-kappa-B pathway (NFKB1) through direct phosphorylation of CARD11/CARMA1 at specific serine residues (Ser-559, Ser-644, and Ser-652) . These phosphorylation events trigger a cascade of molecular interactions leading to immune response regulation, making PRKCB a critical target for immunological research and potentially for therapeutic interventions in immune disorders.

What experimental applications are optimal for PRKCB Antibody, FITC conjugated?

The FITC-conjugated PRKCB antibody demonstrates optimized performance in several research applications. Flow cytometry represents a primary application, as demonstrated in validation studies with Jurkat cells that show clear detection of PRKCB with minimal background signal . For flow cytometry applications, the antibody can be used at concentrations of approximately 1 μg per 10^6 cells. Immunofluorescence (IF) microscopy is another excellent application, with recommended dilutions ranging from 1:50-200 for immunohistochemistry-paraffin (IHC-P) sections . The antibody has also been validated for enzyme-linked immunosorbent assay (ELISA) applications . When designing experiments, researchers should note that while Western blotting is possible with some PRKCB antibodies, the FITC conjugation is particularly advantageous for applications requiring direct visualization without additional detection steps. The observed molecular weight for PRKCB detection is approximately 77 kDa, consistent with the calculated molecular weight of 76.869 kDa . For optimal results in each application, validation with appropriate positive and negative controls is strongly recommended.

What species reactivity has been confirmed for commercial PRKCB antibodies?

Commercial PRKCB antibodies, including FITC-conjugated variants, demonstrate varying species reactivity profiles depending on the specific product and manufacturer. Analysis of available antibodies reveals that many PRKCB antibodies show cross-reactivity with human, mouse, and rat samples . This multi-species reactivity is valuable for comparative studies across different model systems. The FITC-conjugated PRKCB antibody from Assay Genie (PACO55080) specifically reports reactivity with human samples , while the Boster Bio PRKCB antibody (A01940) demonstrates reactivity with human, mouse, and rat samples . Similarly, the VWR antibody targeting both PRKCA and PRKCB shows reactivity with human, mouse, and rat samples . This cross-species reactivity stems from the high conservation of PRKCB protein sequences across mammalian species, particularly in the regions containing the antibody epitopes. When designing experiments involving PRKCB detection in non-human species, researchers should verify the specific cross-reactivity profile of their selected antibody and validate detection in their sample type prior to conducting full-scale experiments.

How can phosphorylation status of PRKCB be effectively monitored using antibody-based detection methods?

Monitoring PRKCB phosphorylation status requires specialized approaches that go beyond total PRKCB detection. While the FITC-conjugated antibodies typically detect total PRKCB regardless of phosphorylation state, phospho-specific PRKCB antibodies are necessary for monitoring activation status. For effective phosphorylation monitoring, researchers should implement a dual-staining approach: using the FITC-conjugated PRKCB antibody to detect total protein levels alongside a separate phospho-specific antibody (conjugated to a spectrally distinct fluorophore) that recognizes specific phosphorylation sites such as Thr500, which is critical for PRKCB activation . Sample preparation requires careful attention to preserve phosphorylation status—samples should be rapidly processed with phosphatase inhibitors included in all buffers. Additionally, stimulation experiments comparing basal versus activated states (e.g., using PMA treatment at 250 ng/mL for 15 minutes) can provide valuable controls for phosphorylation detection . Flow cytometric analysis should include careful compensation when multiple fluorophores are used, and phosphorylation signals should be normalized to total PRKCB expression. Western blot validation of phosphorylation status using the same samples can provide complementary confirmation of the flow cytometry or immunofluorescence results.

What approaches can minimize cross-reactivity with other PKC isoforms when using PRKCB antibodies?

Minimizing cross-reactivity with other PKC isoforms represents a significant challenge when working with PRKCB antibodies due to the high sequence homology between PKC family members. Several approaches can address this challenge: First, select antibodies raised against unique regions of PRKCB—antibodies targeting the C-terminus of PRKCB demonstrate higher specificity compared to those targeting conserved catalytic domains . Second, implement rigorous validation using positive and negative controls, including cell lines with known PRKCB expression patterns and, if possible, PRKCB knockout samples. Third, incorporate peptide blocking experiments as demonstrated in validation studies—when the immunizing peptide blocks antibody signal, this confirms epitope-specific binding . Fourth, use multiple antibodies targeting different epitopes of PRKCB to confirm findings. Fifth, employ isoform-selective PKC inhibitors in functional studies to complement antibody-based detection. Finally, consider using RNA interference (siRNA) to knockdown PRKCB and confirm antibody specificity. The cross-reactivity challenge is particularly important when studying tissues that express multiple PKC isoforms simultaneously, such as brain tissue, where careful optimization of antibody dilution and incubation conditions becomes even more critical.

How does epitope selection influence detection of PRKCB splice variants and protein interactions?

Epitope selection critically influences the detection capabilities of PRKCB antibodies, particularly regarding splice variants and detection of protein complexes. PRKCB exists in two primary splice variants: PRKCB1 and PRKCB2, which differ in their C-terminal regions. Antibodies raised against epitopes in the common regions will detect both variants, while those targeting the unique C-terminal regions offer variant-specific detection . The epitope location relative to functional domains also impacts detection of protein interactions—antibodies targeting interaction interfaces may show reduced binding when PRKCB is engaged in protein complexes. For example, antibodies recognizing epitopes near the CARD11/CARMA1 interaction site might show diminished binding when PRKCB is actively participating in B-cell receptor signaling complexes . Furthermore, post-translational modifications can mask epitopes, leading to variable detection efficiency depending on the activation state of PRKCB. When selecting antibodies for specific research questions, researchers should evaluate whether the epitope is in a conserved region (residues 517-643 for the Assay Genie antibody ) or in regions surrounding specific phosphorylation sites (such as T497 for the Abnova antibody ). For comprehensive analysis, using multiple antibodies targeting different epitopes can provide complementary information about PRKCB conformational states and interaction profiles.

What are the optimal experimental protocols for dual-labeling studies involving PRKCB?

Dual-labeling studies involving FITC-conjugated PRKCB antibodies require careful experimental design to ensure spectral compatibility and minimize artifacts. For successful dual-labeling experiments, first select a compatible second fluorophore with minimal spectral overlap with FITC (e.g., Cy3, Alexa Fluor 594, or APC). When performing immunofluorescence microscopy, follow this optimized protocol: Fix cells using 4% paraformaldehyde for 20 minutes at room temperature, then permeabilize with 0.1% Triton X-100 for 10 minutes . Block with 10% normal goat serum for 30 minutes, then apply the FITC-conjugated PRKCB antibody (1:50-200 dilution) simultaneously with or sequentially after the unconjugated primary antibody for the second target . When performing sequential staining, complete the unconjugated antibody staining with its respective secondary antibody before applying the FITC-conjugated PRKCB antibody. For flow cytometry applications, a similar approach applies but with cell suspensions; fix cells with 4% paraformaldehyde, permeabilize with permeabilization buffer, block with 10% normal goat serum, and stain with the FITC-conjugated PRKCB antibody (1 μg per 10^6 cells) along with the second target antibody . Always include single-stained controls for each fluorophore to enable proper compensation and controls lacking primary antibodies to evaluate background fluorescence. This approach enables simultaneous analysis of PRKCB with interacting partners or cellular markers.

What is the recommended protocol for flow cytometry using PRKCB Antibody, FITC conjugated?

The recommended protocol for flow cytometry using FITC-conjugated PRKCB antibody involves several critical steps to ensure optimal detection. Begin with cell preparation: harvest cells (1-5 × 10^6) and wash twice with cold PBS containing 0.5% BSA. For intracellular staining, fix cells with 4% paraformaldehyde for 15 minutes at room temperature, then permeabilize with permeabilization buffer (0.1% saponin in PBS with 0.5% BSA) for 10 minutes . Next, block non-specific binding by incubating cells with 10% normal goat serum for 30 minutes at room temperature. Apply the FITC-conjugated PRKCB antibody at a concentration of 1 μg per 10^6 cells and incubate for 30 minutes at 20°C in the dark . After incubation, wash cells three times with permeabilization buffer to remove unbound antibody. Resuspend cells in appropriate flow cytometry buffer (PBS with 0.5% BSA and 2 mM EDTA) for analysis. Critical controls should include: (1) unstained cells, (2) cells stained with isotype control antibody (rabbit IgG-FITC), and (3) cells processed without primary antibody incubation but with all other steps identical . During analysis, establish appropriate gates based on forward and side scatter to exclude cell debris and aggregates, and set fluorescence thresholds using the control samples. This protocol typically yields a single peak shift for PRKCB-positive populations compared to the negative controls when visualized on histogram plots.

What optimization steps are required for immunohistochemistry with PRKCB antibodies?

Optimizing immunohistochemistry (IHC) with PRKCB antibodies requires systematic adjustment of several parameters to achieve specific staining with minimal background. The following optimization protocol is recommended: For paraffin-embedded tissues, begin with deparaffinization and rehydration through xylene and graded alcohols. Antigen retrieval is critical—heat-mediated antigen retrieval in EDTA buffer (pH 8.0) provides optimal epitope exposure for PRKCB detection . Following antigen retrieval, block endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes if using HRP-based detection systems. Next, block non-specific binding with 10% goat serum for 30 minutes at room temperature. Apply the PRKCB antibody at various dilutions (starting with 1:50-200 for IF/IHC-P) to identify optimal concentration . For FITC-conjugated antibodies, protect slides from light during and after antibody incubation. Incubate primary antibody overnight at 4°C in a humidified chamber for optimal results. For non-conjugated antibodies, apply appropriate secondary antibody (e.g., Peroxidase Conjugated Goat Anti-rabbit IgG) for 30 minutes at 37°C. Develop signal using DAB for chromogenic detection or proceed directly to counterstaining and mounting for fluorescent detection . Optimization variables include antibody concentration, incubation time/temperature, antigen retrieval method, and blocking conditions. Always include positive control tissues (e.g., brain tissue for PRKCB) and negative controls (primary antibody omitted) in optimization experiments.

What troubleshooting approaches address common challenges with PRKCB antibody experiments?

When encountering challenges with PRKCB antibody experiments, systematic troubleshooting approaches can resolve most issues. For weak or absent signals, first verify PRKCB expression in your sample type through literature or database searches. Increase antibody concentration (reducing dilution factor) or extend incubation time (from 1 hour to overnight at 4°C). Optimize antigen retrieval—EDTA buffer (pH 8.0) has shown superior results for PRKCB detection compared to citrate buffer . For high background or non-specific staining, increase blocking stringency using 10% serum from the same species as the secondary antibody, add 0.1-0.3% Triton X-100 to reduce non-specific binding, and ensure thorough washing between steps (3-5 washes, 5 minutes each). When experiencing cross-reactivity with other PKC isoforms, perform peptide competition assays with the immunizing peptide to confirm specificity . For fluorescence applications with photobleaching concerns, minimize exposure to light during all steps, use antifade mounting medium, and consider dipping slides in 0.1% Sudan Black in 70% ethanol before mounting to reduce autofluorescence. If inconsistent results occur between experiments, standardize all protocols including sample preparation, fixation duration, buffer compositions, and implement detailed record-keeping for all experimental parameters. Finally, for degraded epitopes, ensure samples are processed quickly after collection and include protease inhibitors in all buffers when working with fresh tissues.

How can PRKCB activation be experimentally induced and monitored?

PRKCB activation can be experimentally induced and monitored through a combination of pharmacological approaches and detection methods. To induce PRKCB activation, researchers commonly use phorbol esters such as PMA (phorbol 12-myristate 13-acetate) at concentrations of 250 ng/mL for 15 minutes, which directly activates PKC isoforms by mimicking diacylglycerol . Alternative physiological activation can be achieved through B-cell receptor crosslinking using anti-IgM antibodies in B lymphocytes, or through treatment with calcium ionophores combined with diacylglycerol analogs. For monitoring activation, several complementary approaches are recommended: (1) Western blot analysis using phospho-specific antibodies targeting activation-associated phosphorylation sites, (2) translocation assays monitoring PRKCB movement from cytosol to membrane using fractionation or live-cell imaging with fluorescently tagged PRKCB, (3) kinase activity assays using PRKCB-specific substrates, and (4) flow cytometry with phospho-specific antibodies to quantify activation at the single-cell level . When designing activation experiments, time course analyses (0, 5, 15, 30, 60 minutes) provide valuable insights into activation kinetics. Inhibitor controls using PKC-specific inhibitors (e.g., Gö6976 for classical PKCs) confirm the specificity of observed effects. This multi-parametric approach to monitoring PRKCB activation provides comprehensive understanding of its regulation and function in various cellular contexts.

What validation criteria should be applied to PRKCB antibodies before experimental use?

Before experimental use, PRKCB antibodies should undergo rigorous validation according to established criteria to ensure reliability and reproducibility. The recommended validation workflow includes several key steps: First, perform Western blot analysis to confirm the antibody detects a single band of the expected molecular weight (approximately 77 kDa for PRKCB) . Second, conduct peptide competition assays where pre-incubation of the antibody with the immunizing peptide should abolish specific staining, as demonstrated in validation images for commercial antibodies . Third, validate specificity across multiple applications relevant to your research (e.g., Western blot, IHC, IF, flow cytometry) to confirm consistent performance. Fourth, test antibody performance in positive control samples known to express PRKCB (e.g., Jurkat cells, brain tissue, B lymphocytes) and negative control samples with minimal PRKCB expression . Fifth, evaluate lot-to-lot consistency when receiving new antibody batches by comparing performance with previously validated lots. Sixth, if available, test antibody performance in knockdown/knockout systems to definitively confirm specificity. Finally, cross-validate findings with alternative antibodies targeting different epitopes of PRKCB. Documentation of these validation steps, including images and methodological details, should be maintained as part of laboratory records to support the reliability of subsequent experimental results.

What reference standards and controls are essential for experiments using FITC-conjugated PRKCB antibodies?

Rigorous experimental design with FITC-conjugated PRKCB antibodies requires comprehensive controls and reference standards to ensure valid and interpretable results. Essential controls include: (1) Isotype controls—rabbit IgG-FITC antibodies at matching concentrations to assess non-specific binding (e.g., 1 μg per 10^6 cells for flow cytometry) ; (2) Unstained/blank controls—samples processed identically but without primary antibody to establish autofluorescence baseline; (3) Positive tissue/cell controls—samples with established PRKCB expression such as brain tissue or Jurkat cells ; (4) Negative tissue/cell controls—samples with minimal PRKCB expression; (5) Peptide competition controls—antibody pre-incubated with immunizing peptide to confirm binding specificity ; and (6) Stimulation controls—paired samples with and without PKC activators (e.g., PMA treatment at 250 ng/mL for 15 minutes) to demonstrate detection of activation-dependent changes . For fluorescence microscopy and flow cytometry, single-color controls are necessary for spectral compensation when performing multi-parameter analyses. Additionally, standardized beads with defined fluorescence intensities serve as reference standards for quantitative applications, enabling comparison between experiments and instruments. These comprehensive controls should be incorporated into experimental design and data analysis workflows to ensure scientific rigor and reproducibility when working with FITC-conjugated PRKCB antibodies.

How can PRKCB antibody performance be quantitatively assessed across experimental applications?

Quantitative assessment of PRKCB antibody performance requires systematic evaluation across multiple parameters and applications. For Western blot applications, quantitative metrics include signal-to-noise ratio (optimally >10:1), linear dynamic range (typically 2-3 orders of magnitude for chemiluminescence detection), and consistency of detection across a protein concentration gradient. For immunofluorescence applications, performance metrics include coefficient of variation between replicate measurements (<15% for robust antibodies), background fluorescence intensity in negative control regions (optimally <10% of specific signal), and resolution of subcellular localization patterns. For flow cytometry, key metrics include stain index (mean positive signal minus mean negative signal, divided by twice the standard deviation of the negative population), with values >50 indicating excellent performance for FITC-conjugated antibodies . Performance assessment should also include reproducibility measurements across multiple experiments and operators, with coefficient of variation ideally <20%. Antibody titration experiments can define the optimal concentration that yields maximum specific signal with minimal background across applications (for IHC-P, recommended dilutions typically range from 1:50-200) . Sensitivity can be quantitatively assessed using recombinant PRKCB protein standards at defined concentrations. These quantitative assessments provide objective criteria for antibody selection and experimental optimization while enabling meaningful comparison between different commercial PRKCB antibodies.

How can PRKCB antibodies be integrated into high-content imaging workflows?

Integration of FITC-conjugated PRKCB antibodies into high-content imaging workflows enables comprehensive spatial and quantitative analysis of PRKCB expression, localization, and activation states at scale. An optimized workflow begins with standardized sample preparation on imaging-compatible plates, followed by automated immunostaining using liquid handling systems to ensure consistency. For multiplex imaging, combine FITC-conjugated PRKCB antibody with spectrally distinct markers for cell compartments (nuclear, cytoplasmic, membrane) and interaction partners . Automated image acquisition using high-content systems should capture multiple fields per well at appropriate magnification (40-60×) to resolve subcellular localization. Image analysis pipelines should incorporate: (1) nuclear identification using DAPI or Hoechst staining, (2) cell boundary delineation using cytoplasmic markers, (3) background correction algorithms, (4) intensity quantification in defined cellular compartments, and (5) measurement of PRKCB translocation using nuclear/cytoplasmic or membrane/cytoplasmic intensity ratios. Advanced analysis can include correlation of PRKCB distribution with morphological parameters and co-localization with interaction partners. Quality control metrics should assess Z' factors between positive and negative controls (optimally >0.5) and coefficient of variation between replicates (<15%). This integrated high-content approach enables screening of compounds affecting PRKCB localization and activation, assessment of PRKCB dynamics in heterogeneous cell populations, and quantification of subtle phenotypic changes associated with PRKCB modulation.

What emerging technologies complement antibody-based detection of PRKCB in research applications?

Several emerging technologies complement and extend traditional antibody-based PRKCB detection, offering new insights into its biology and function. CRISPR-Cas9 genome editing for endogenous tagging of PRKCB with fluorescent proteins or small epitope tags enables live-cell imaging of dynamics without antibody limitations. Proximity ligation assays (PLA) using PRKCB antibodies paired with antibodies against interaction partners provide superior sensitivity for detecting protein-protein interactions, generating punctate fluorescent signals only when proteins are within 40 nm proximity . Mass cytometry (CyTOF) using metal-conjugated PRKCB antibodies enables highly multiplexed single-cell analysis without spectral overlap concerns. Single-molecule localization microscopy (SMLM) techniques like STORM or PALM with fluorophore-conjugated PRKCB antibodies achieve super-resolution imaging below the diffraction limit (<50 nm), revealing nanoscale organization. RNA-protein co-detection methods combining PRKCB antibody staining with RNA FISH allow simultaneous visualization of protein expression and mRNA abundance. Spatial transcriptomics technologies integrating antibody detection with location-resolved RNA sequencing provide comprehensive tissue-wide expression mapping. Optogenetic approaches using light-controllable PKC variants complement antibody studies by enabling precise spatiotemporal control of PRKCB activation. These technologies, when combined with traditional antibody-based methods, provide multi-dimensional insights into PRKCB biology across scales from molecules to tissues, advancing our understanding of its roles in normal physiology and disease states.