PPP6C Antibody, FITC conjugated

What is PPP6C and what cellular functions make it a significant research target?

PPP6C (Protein Phosphatase 6, Catalytic Subunit) functions as the catalytic subunit of protein phosphatase 6 (PP6). It plays critical roles in multiple cellular pathways, making it a valuable research target. PPP6C is involved in regulating cell cycle progression in response to IL2 receptor stimulation, with its N-terminal domain specifically restricting G1 to S phase progression in cancer cells partly through cyclin D1 control . During mitosis, PPP6C regulates spindle positioning, ensuring proper chromosomal segregation .

Additionally, PPP6C participates in immune signaling by downregulating MAP3K7 kinase activation in the IL1 signaling pathway through MAP3K7 dephosphorylation . Importantly, PPP6C is a key participant in innate immune defense against viruses by dephosphorylating RIG-I/DDX58, which is essential for triggering RIG-I/DDX58-mediated signaling activation . Recent research has also revealed that PPP6C negatively regulates the cGAS-STING pathway by removing STING phosphorylation required for its activation .

How does FITC conjugation affect PPP6C antibody performance in immunofluorescence applications?

FITC (Fluorescein isothiocyanate) conjugation converts the PPP6C antibody into a directly detectable reagent, eliminating the need for secondary antibodies in immunofluorescence studies. The conjugation process attaches the FITC fluorophore to the antibody molecule, typically at lysine residues, without significantly affecting the antibody's binding properties when properly performed.

When working with FITC-conjugated PPP6C antibodies, researchers should account for the following technical considerations:

• FITC has an excitation maximum at approximately 495 nm and emission maximum at around 519 nm, requiring appropriate filter sets

• FITC is susceptible to photobleaching, necessitating anti-fade mounting media and minimized exposure to excitation light

• The fluorophore's quantum yield may be affected by environmental pH, with optimal performance at slightly alkaline conditions (pH 8.0-9.0)

Multiple validated applications for FITC-conjugated PPP6C antibodies include immunofluorescence (IF), as confirmed by product specifications from commercial suppliers .

What sample preparation techniques maximize signal-to-noise ratio when using PPP6C antibody, FITC conjugated?

Optimizing sample preparation is critical for achieving high signal-to-noise ratios with FITC-conjugated PPP6C antibodies. The following methodological approach is recommended:

Fixation optimization:

• For intracellular antigens like PPP6C, use 4% paraformaldehyde for 15-20 minutes at room temperature

• For membrane preservation, consider combining with 0.1% glutaraldehyde

• Assess whether methanol fixation (-20°C for 10 minutes) might better preserve PPP6C epitopes in your specific cell type

Permeabilization considerations:

• Use 0.1-0.3% Triton X-100 in PBS for 10 minutes for nuclear and cytoplasmic PPP6C

• For delicate structures, substitute with 0.1% saponin, which maintains reversible permeabilization

Blocking protocol:

• Block with 5-10% normal serum from a species different from the antibody's host (rabbit for most PPP6C antibodies)

• Include 0.1-0.3% Triton X-100 and 1% BSA in blocking buffer

• Extend blocking time to 1-2 hours at room temperature to reduce non-specific binding

Antibody incubation:

• Dilute FITC-conjugated PPP6C antibody in blocking buffer at experimentally determined optimal concentration

• Incubate overnight at 4°C in a humidified chamber protected from light

• Include appropriate controls, including isotype-matched FITC-conjugated non-specific antibodies

Rigorous washing steps between each stage with PBS containing 0.05% Tween-20 significantly improves signal-to-noise ratio in final images.

How can PPP6C antibody, FITC conjugated be utilized to investigate its role in the cGAS-STING pathway?

Investigating PPP6C's role in the cGAS-STING pathway using FITC-conjugated antibodies requires sophisticated experimental approaches that integrate immunofluorescence with functional assays. Recent research has established that PPP6C negatively regulates STING-dependent innate immune responses by removing STING phosphorylation, which is required for pathway activation .

Suggested experimental workflow:

• Co-localization studies:

  • Perform dual immunofluorescence using FITC-conjugated PPP6C antibody and a complementary fluorophore-conjugated STING antibody

  • Analyze co-localization before and after pathway stimulation with cytosolic DNA or cGAMP

  • Quantify Pearson's correlation coefficient or Manders' overlap coefficient across different timepoints post-stimulation

• Phosphorylation dynamics analysis:

  • Combine FITC-conjugated PPP6C antibody with phospho-specific STING antibodies to track the relationship between PPP6C localization and STING phosphorylation status

  • Use phosphatase inhibitors (calyculin A or okadaic acid) as controls to confirm the specificity of PPP6C's effect on STING phosphorylation

• Translocation assessment:

  • Track PPP6C recruitment to STING-containing compartments following pathway activation

  • Document temporal changes in PPP6C localization relative to STING trafficking from ER to Golgi

Research data indicates that knockdown of PPP6C significantly increases dsDNA- and cGAMP-induced IFN-β production compared to control cells, providing a functional readout to correlate with imaging results . Importantly, while phosphorylation of TBK1 in PPP6C-depleted cells shows minimal change, phosphorylation of IRF3 is greatly increased, suggesting that visualizing PPP6C-IRF3 interactions may provide mechanistic insights .

What considerations should be made when using PPP6C antibody, FITC conjugated for visualization of virus-host interactions?

Visualizing PPP6C's role in virus-host interactions using FITC-conjugated antibodies requires careful experimental design that accounts for both viral dynamics and PPP6C's regulatory functions. Research has demonstrated that PPP6C deficiency enhances HSV-1-induced IFN-β production and inhibits viral replication .

Critical methodology considerations:

• Timing of fixation and analysis:

  • Establish a time-course experiment capturing early (0-2h), middle (4-8h), and late (12-24h) stages of viral infection

  • Correlate PPP6C localization with viral replication compartments at each timepoint

  • Use sub-lethal viral doses to prevent cytopathic effects that compromise cellular architecture

• Viral strain selection:

  • Utilize fluorescently-tagged reporter viruses (different emission spectrum from FITC) for simultaneous visualization

  • For HSV-1 studies, consider ICP0-null mutants which differentially engage innate immune sensing

• Control experimental conditions:

  • Include non-infected controls at matched timepoints

  • Compare wild-type virus with immune-evasion deficient strains

  • Use selective inhibitors of PPP6C to confirm phenotypes observed in siRNA experiments

• Data analysis approach:

  • Quantify nuclear-cytoplasmic ratios of PPP6C during infection progression

  • Measure co-localization coefficients between PPP6C and viral components

  • Correlate PPP6C redistribution with markers of innate immune activation

Experimental evidence shows that PPP6C deficiency enhances both HSV-1 (DNA virus) and VSV (RNA virus) induced IFN-β production while inhibiting viral replication, suggesting a conserved regulatory role across different viral infections . The enhancement of IRF3 phosphorylation in PPP6C-deficient cells during viral infection provides a measurable output to correlate with imaging data .

How can quantitative immunofluorescence be optimized to measure PPP6C-mediated dephosphorylation of target proteins?

Measuring PPP6C-mediated dephosphorylation events through quantitative immunofluorescence requires rigorous technical optimization to generate reliable and reproducible data. The following methodology addresses key considerations:

Technical setup for quantitative measurements:

• Multiplex immunofluorescence approach:

  • Use FITC-conjugated PPP6C antibody in combination with far-red fluorophore-conjugated phospho-specific antibodies against known targets

  • Include nuclear counterstain (DAPI) for segmentation and normalization purposes

  • Maintain identical acquisition parameters across experimental conditions

• Calibration standards:

  • Include calibration slides with known fluorophore concentrations in each experiment

  • Generate standard curves to convert fluorescence intensity to molecular equivalents of soluble fluorophore (MESF)

  • Use biological standards (cells with defined PPP6C expression levels) as inter-experimental controls

• Image acquisition parameters:

  • Determine linear dynamic range of your detection system before experiments

  • Ensure exposure settings prevent pixel saturation while maximizing signal

  • Maintain consistent microscope settings, including gain, offset, and laser power

Experimental design for phosphatase activity assessment:

Research data demonstrates that knockdown of PPP6C markedly enhances dsDNA-induced phosphorylation of IRF3 without significantly affecting TBK1 phosphorylation, providing valuable targets for quantitative analysis .

What are common technical issues encountered with PPP6C antibody, FITC conjugated and their solutions?

Researchers frequently encounter technical challenges when working with FITC-conjugated PPP6C antibodies. The following troubleshooting guide addresses these issues with methodological solutions:

Low signal intensity:

• Cause: Insufficient permeabilization limiting antibody access to intracellular PPP6C

• Solution: Optimize permeabilization by testing increasing concentrations of detergent (0.1% to 0.5% Triton X-100) or using alternative permeabilization agents like digitonin for membrane proteins

High background fluorescence:

• Cause: Inadequate blocking or excessive antibody concentration

• Solution: Extend blocking time to 2 hours, increase blocking agent concentration to 10%, and titrate antibody to determine optimal concentration using a checkerboard titration approach

Photobleaching during acquisition:

• Cause: FITC susceptibility to photobleaching under prolonged excitation

• Solution: Use anti-fade mounting media containing radical scavengers, minimize exposure time, and consider time-resolved acquisition when tracking dynamic processes

Inconsistent staining patterns:

• Cause: Variability in fixation affecting epitope accessibility

• Solution: Standardize fixation protocol with precise timing and temperature control; consider comparing multiple fixation methods (paraformaldehyde, methanol, or hybrid approaches)

Non-specific nuclear staining:

• Cause: Charge-based interactions between FITC-conjugated antibodies and nucleic acids

• Solution: Increase salt concentration in wash buffers (up to 500 mM NaCl) and include 0.05% Tween-20 in all wash steps

Autofluorescence interference:

• Cause: Natural cellular autofluorescence in the FITC emission spectrum

• Solution: Implement spectral unmixing during image acquisition or use Sudan Black B (0.1% in 70% ethanol) treatment post-staining to quench autofluorescence

Research validation methods confirm that PPP6C antibodies effectively detect the target protein, with several antibodies demonstrating reactivity across human, mouse, and rat samples in applications including ELISA, Western blot (WB), immunohistochemistry (IHC), and immunofluorescence (IF) .

How can contradictory data regarding PPP6C function be resolved through advanced imaging techniques?

Resolving contradictory findings regarding PPP6C function requires sophisticated imaging approaches that can distinguish between different protein complexes, phosphorylation states, and subcellular localizations. The following methodological framework addresses this challenge:

Advanced microscopy techniques for resolution enhancement:

• Super-resolution microscopy:

  • Implement Structured Illumination Microscopy (SIM) to achieve ~120 nm resolution

  • Apply Stimulated Emission Depletion (STED) microscopy for visualizing PPP6C-STING interactions below the diffraction limit

  • Use Single Molecule Localization Microscopy (PALM/STORM) for nanoscale distribution analysis

• Proximity-based interaction assessment:

  • Employ Förster Resonance Energy Transfer (FRET) between FITC-labeled PPP6C and acceptor fluorophore-labeled interaction partners

  • Implement Proximity Ligation Assay (PLA) to verify protein-protein interactions with <40 nm proximity

  • Utilize split-fluorescent protein complementation assays to confirm direct interactions

• Live-cell temporal analysis:

  • Correlate FITC-PPP6C antibody staining patterns with live-cell experiments using fluorescent protein-tagged PPP6C

  • Perform Fluorescence Recovery After Photobleaching (FRAP) to measure protein dynamics

  • Implement optogenetic approaches to spatiotemporally control PPP6C activity

Research application examples:

Similarly, the temporal dynamics of PPP6C's involvement in antiviral responses can be comprehensively mapped using time-resolved imaging. Research data indicates that the enhancement of IFN-β production in PPP6C-depleted cells is maintained across various time points after dsDNA stimulation but follows different patterns with other stimuli like poly(I:C) .

How can PPP6C antibody, FITC conjugated be used in multi-parameter analyses of phosphorylation networks?

Multi-parameter analysis of phosphorylation networks using FITC-conjugated PPP6C antibodies enables comprehensive mapping of signaling dynamics in complex cellular systems. The methodological approach below integrates various techniques to achieve high-dimensional data:

Technical implementation:

• Multiplexed immunofluorescence strategy:

  • Combine FITC-conjugated PPP6C antibody with spectrally distinct fluorophore-conjugated antibodies against phosphorylated substrates

  • Implement cyclic immunofluorescence with antibody stripping and reprobing to measure >10 parameters on the same sample

  • Use spectral unmixing algorithms to separate overlapping emission spectra

• Single-cell analysis pipeline:

  • Segment cells using nuclear and cytoplasmic markers

  • Extract intensity features from multiple channels on a per-cell basis

  • Apply dimensionality reduction techniques (tSNE, UMAP) to identify cell subpopulations

  • Perform trajectory analysis to map temporal evolution of phosphorylation states

• Integrative data analysis:

  • Correlate imaging data with parallel biochemical assays (phospho-proteomics)

  • Apply machine learning algorithms to identify predictive phosphorylation patterns

  • Construct computational models of phosphorylation/dephosphorylation kinetics

Research-based protocol design:

Research evidence indicates that PPP6C functions within complex signaling networks. For example, in antiviral immunity, PPP6C negatively regulates dsDNA-induced IRF3 activation but not NF-κB activation . This differential regulation highlights the importance of multi-parameter analysis to fully characterize phosphatase substrate specificity.