PRKCA Antibody, FITC conjugated

What is PRKCA and why is it important in cellular signaling research?

PRKCA (Protein Kinase C Alpha) is a 77 kDa protein involved in cAMP-dependent signaling triggered by receptor binding to GPCRs. PKC activation regulates diverse cellular processes including:

• Cell proliferation and differentiation

• Cell cycle progression

• Microtubule dynamics

• Chromatin condensation and decondensation

• Nuclear envelope assembly/disassembly

• Intracellular transport mechanisms

• Ion flux regulation

PRKCA exists in active and inactive conformations, with the active form typically associated with membrane localization following stimuli like PMA (phorbol 12-myristate 13-acetate) treatment. The protein shuttles between cytoplasmic and membrane compartments during activation/deactivation cycles, making it a critical subject for dynamic cellular signaling studies .

What are the key applications for FITC-conjugated PRKCA antibodies in research?

FITC-conjugated PRKCA antibodies are utilized across multiple experimental platforms:

Flow cytometry applications are particularly well-suited for FITC-conjugated antibodies, as demonstrated by studies analyzing PRKCA expression in peripheral blood lymphocytes and various cell lines including HeLa and Jurkat cells .

How should samples be prepared for flow cytometry using PRKCA-FITC antibodies?

For optimal flow cytometry results with PRKCA-FITC antibodies:

• Harvest cells (1×10^6 cells per sample) and wash twice with PBS

• Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilize cells with 0.1% Triton X-100 or commercial permeabilization buffer for 5-10 minutes

• Block with 5% BSA in PBS for 30 minutes

• Incubate with FITC-conjugated PRKCA antibody (typically 0.4 μg per 10^6 cells in 100 μl suspension)

• Wash 3 times with PBS before flow cytometric analysis

Research data shows successful application in peripheral blood lymphocytes where cells were fixed, permeabilized and stained with anti-PRKCA FITC (10 μl per test) alongside appropriate isotype controls . Cell preparation requires permeabilization as PRKCA is primarily an intracellular target.

What are the optimal dilution ratios for PRKCA-FITC antibodies across different applications?

Based on experimental validation across multiple studies, the following dilution ratios are recommended:

It is recommended to titrate these antibodies in each testing system to obtain optimal results, as sensitivity can vary by sample type and experimental conditions . Some protocols specifically recommend IF(IHC-P) dilutions at 1:50-200 for certain antibody clones .

How can researchers distinguish between active and inactive PRKCA using antibody-based approaches?

Distinguishing active from inactive PRKCA requires specific considerations:

• Conformational antibodies: Some antibodies (like anti-C2Cat) are specifically designed to recognize the active conformation of PRKCA. These antibodies bind epitopes exposed only when PRKCA adopts its active configuration.

• Phosphorylation-specific antibodies: Antibodies targeting phosphorylated residues (such as phospho-Ser657/Tyr658) detect activated PRKCA since phosphorylation is often associated with activation.

• Subcellular fractionation approach: Active PRKCA translocates to the membrane. Researchers can:

  • Separate cytosolic and membrane fractions

  • Probe fractions with PRKCA antibodies

  • Quantify membrane/cytosol ratio as an activation indicator

Studies have validated this approach using wild-type PKC versus N-terminally truncated PKC (ΔNPSPKCβΙ without the first 30 amino acids where the pseudo-substrate site is located). The truncated form adopts a permanently active conformation and shows increased membrane localization regardless of PMA treatment .

What controls should be included when using PRKCA-FITC antibodies in experiments?

Proper experimental controls are essential for reliable data interpretation:

Flow cytometry experiments specifically benefit from using isotype controls to establish proper gating strategies. Published data demonstrates how blood cells from healthy patients stained with anti-PKCα FITC (blue signal) compared to isotype control (black signal) can clearly distinguish specific from non-specific staining .

What are common challenges in PRKCA-FITC signal detection and how can they be addressed?

Several technical challenges may arise when working with PRKCA-FITC antibodies:

• High background fluorescence:

  • Ensure proper blocking (5% BSA or serum for 30-60 minutes)

  • Increase washing steps (3-5 times with PBS containing 0.05% Tween-20)

  • Optimize antibody dilution through titration experiments

  • Use freshly prepared fixation solutions

• Weak or absent signal:

  • Verify sample preparation (proper fixation and permeabilization)

  • Check antibody storage conditions (many require storage at 4°C with 50% glycerol)

  • Confirm PRKCA expression in your cell type/tissue

  • Try antigen retrieval for tissue sections (TE buffer pH 9.0 or citrate buffer pH 6.0)

• Inconsistent results:

  • Standardize protocols (cell number, incubation time, temperature)

  • Use freshly prepared samples

  • Ensure consistent instrument settings for flow cytometry or microscopy

Research data indicates that cell-specific optimization is necessary, as successful staining has been documented in HeLa, NIH/3T3, HepG2, and Jurkat cells, but conditions may vary between cell types .

How can temporal dynamics of PRKCA activation be monitored using FITC-conjugated antibodies?

PRKCA activation dynamics can be monitored through several approaches:

• Time-course experiments:

  • Treat cells with activators (PMA, ATP, glutamate) for various durations

  • Fix cells at specific timepoints

  • Analyze active PRKCA using conformation-specific antibodies

• Live-cell imaging:

  • Use cell-permeable fluorescent PKC activators alongside FITC-antibodies in fixed timepoints

  • Track membrane translocation events

• Pulse-chase activation:

  • Pulse with activator (e.g., 100 nM PMA, 1 μM ATP, or 1 μM glutamate)

  • Chase for different durations

  • Fix and analyze PRKCA distribution

Published research demonstrates that PRKCA activation by PMA is sustained up to 30 minutes, while receptor-mediated activation by ATP peaks at 1 minute and glutamate at 3 minutes of treatment. This approach has been validated in SK-N-SH cells using immunofluorescence with conformation-specific antibodies, revealing that receptor-mediated activation is typically rapid and transient compared to PMA-induced activation .

How does DNA methylation influence PRKCA expression and what methods can detect this regulation?

Recent research has uncovered a novel regulatory mechanism involving DNA methylation and the long non-coding RNA PRKCA-AS1:

• DNA methylation regulation of PRKCA:

  • Methylation of PRKCA promoter and first exon regions negatively correlates with expression

  • DNMT1 (DNA methyltransferase 1) particularly influences PRKCA expression

  • TNF-α treatment decreases methylation and increases PRKCA expression

• Detection methods:

  • Bisulfite sequencing to quantify DNA methylation levels

  • ChIP assays to assess DNMT binding to PRKCA regulatory regions

  • DNMT knockdown experiments to establish causality

  • 5-Azacytidine (DNMTs inhibitor) treatment as a positive control

Research data shows that PRKCA is upregulated after TNF-α treatment (FC = 3.23, p = 0.037 in AC16 cells; FC = 2.96, p = 0.04 in RL-14 cells), DNMT1 deficiency (FC = 3.7, p = 0.031 in AC16; FC = 2.74, p = 0.047 in RL-14), or 5-Azacytidine addition (FC = 3.57, p = 0.034 in AC16; FC = 2.89, p = 0.042 in RL-14). These treatments also reduced DNA methylation levels in the promoter and first exon regions .

What is the role of PRKCA-AS1 in regulating PRKCA and how can this interaction be studied?

PRKCA-AS1 is a long non-coding RNA that interacts with PRKCA regulation:

• Interaction mechanism:

  • PRKCA-AS1 binds to PRKCA through its 5' terminal (first exon)

  • The binding may influence DNA methylation patterns at the PRKCA locus

  • Computational analysis suggests PRKCA-AS1 may target the PRKCA promoter

• Experimental approaches:

  • RNA immunoprecipitation (RIP) to detect RNA-protein interactions

  • Truncation experiments with different PRKCA-AS1 exons to map binding sites

  • Computational tools like "Triplex Domain Finder" for predicting genomic targets

Studies have shown that when truncated versions of PRKCA-AS1 lacking the first exon were transfected into AC16 cells, the binding affinity for PRKCA was lost, confirming that the 5' terminal of PRKCA-AS1 is essential for PRKCA binding .

How can researchers effectively measure changes in PRKCA activation in response to inflammatory stimuli?

Inflammatory activation of PRKCA can be measured through several complementary approaches:

• Cell culture models:

  • Treat cardiomyocyte cell lines (like AC16 and RL-14) with inflammatory cytokines (e.g., 100 ng/mL TNF-α)

  • Validate inflammatory response through ELISA for IL-1β and IL-4 in culture medium

  • Assess PRKCA expression and activation state

• Protein dynamics analysis:

  • Western blotting with phospho-specific PRKCA antibodies

  • Membrane fractionation to assess translocation

  • Immunofluorescence to visualize subcellular redistribution

• Quantification methods:

  • Flow cytometry with FITC-conjugated antibodies for population analysis

  • Image analysis of confocal microscopy data to quantify membrane/cytosol ratios

  • Co-localization with membrane markers

Research has established that inflammatory conditions significantly alter both PRKCA expression levels and its activation state, making these approaches valuable for studying inflammatory signaling cascades .

What are the optimal storage conditions for FITC-conjugated PRKCA antibodies?

Proper storage is critical for maintaining antibody functionality:

Multiple manufacturers recommend storing FITC-conjugated antibodies at 4°C for up to 12 months, or at -20°C/-80°C for longer periods, while emphasizing the importance of avoiding repeated freezing and thawing .

How can researchers verify the integrity of FITC-conjugated antibodies before experimental use?

Before conducting critical experiments, verify antibody integrity through:

• Positive control testing:

  • Test on validated cell lines known to express PRKCA (HeLa, Jurkat, NIH/3T3)

  • Compare signal intensity to previous lots or reference standards

• Spectroscopic assessment:

  • Measure absorption/emission spectra

  • FITC should show characteristic absorption at ~495 nm and emission at ~520 nm

  • Protein:dye ratio can indicate conjugation efficiency

• Functionality tests:

  • Small-scale flow cytometry run with positive control cells

  • Microscopy check on fixed control samples

  • Western blot verification of expected 77 kDa band