PPP6C Antibody, Biotin conjugated

What is PPP6C and what cellular functions does it regulate?

PPP6C (protein phosphatase 6 catalytic subunit) functions as the catalytic component of the PP6 holoenzyme complex. It plays crucial roles in multiple cellular pathways including cell cycle progression, mitotic regulation, and immune responses. Specifically, PPP6C regulates G1 to S phase progression in cancer cells through control of cyclin D1 and governs spindle positioning during mitosis . It also serves as a key negative regulator in innate immunity by dephosphorylating and inactivating components of the cGAS-STING pathway, including dephosphorylation of CGAS at Ser-435 which impairs its GTP-binding capability . Additionally, PPP6C down-regulates MAP3K7 kinase activation in the IL1 signaling pathway through direct dephosphorylation .

How does PPP6C holoenzyme assembly influence antibody selection?

PPP6C does not function in isolation but forms complexes with regulatory subunits including PP6R1-3, ANKRD28, ANKRD44, ANKRD52, and interacts with proteins such as IGBP1 and TIPRL . When selecting a PPP6C antibody, consider the epitope location and whether it might be masked when PPP6C is incorporated into these protein complexes. Antibodies targeting the C-terminal region (such as those generated against the NH2-Cys-Ala-Val-Pro-Asp-Ser-Glu-Arg-Val-Ile-Pro-Pro-Arg-Thr-Thr-Thr-Pro-Tyr-COOH peptide) may detect PPP6C regardless of its complex formation state, while those targeting regions involved in protein-protein interactions may only detect free PPP6C.

What species reactivity should be considered when selecting a PPP6C antibody?

Available PPP6C antibodies demonstrate varying species reactivity profiles. Some antibodies show broad cross-reactivity across human, mouse, rat, and bovine samples , while others are specifically optimized for human samples . When planning experiments involving multiple species or using animal models, select antibodies with confirmed cross-reactivity or validate the antibody in your specific species of interest. The high degree of sequence conservation in PPP6C across mammalian species often enables cross-reactivity, but experimental validation remains essential.

What validation approaches confirm PPP6C antibody specificity?

Rigorous validation of PPP6C antibody specificity is critical for reliable research outcomes. A comprehensive validation approach includes:

• Western blot analysis demonstrating a single band at the expected molecular weight (approximately 35 kDa for native PPP6C or 42 kDa for tagged variants)

• Peptide competition assays showing signal elimination when the antibody is pre-incubated with the immunizing peptide

• Genetic validation using cells with PPP6C knockdown, knockout, or degradation systems

• Mass spectrometry confirmation of antibody-enriched proteins to verify target specificity

Research has demonstrated successful validation through band shift assays comparing wild-type PPP6C (35 kDa) with tagged versions (42 kDa), confirming antibody specificity to all endogenously expressed PPP6C protein .

How should biotin-conjugated PPP6C antibodies be optimized for ELISA applications?

For optimal ELISA performance with biotin-conjugated PPP6C antibodies:

• Concentration optimization: Perform titration experiments using 0.1-10 μg/ml ranges to determine optimal signal-to-noise ratios

• Blocking optimization: Test multiple blocking agents (BSA, casein, normal serum) to minimize background

• Amplification strategy: Utilize streptavidin-HRP conjugates with enhanced sensitivity substrates like TMB Ultra for improved detection

• Capture antibody pairing: When designing sandwich ELISAs, pair the biotin-conjugated detection antibody with a capture antibody targeting a distinct PPP6C epitope (amino acids 40-211 versus C-terminal epitopes)

• Sample preparation: Consider phosphatase inhibitors in lysis buffers to preserve the native phosphorylation state of PPP6C

The biotin-conjugated polyclonal antibodies generated against recombinant human PPP6C (amino acids 40-211) offer optimal performance for detecting human PPP6C in complex samples .

What are the critical considerations for investigating PPP6C's role in phosphorylation dynamics?

When studying PPP6C's phosphatase activity and its impact on substrate phosphorylation:

• Establish baseline phosphorylation: Quantify the phosphorylation state of known PPP6C substrates (e.g., Aurora A at T288, Tastin at S156, NDC80 at S55) under normal conditions

• Temporal analysis: Design time-course experiments to capture dynamic phosphorylation changes following PPP6C manipulation

• Inhibitor controls: Include phosphatase inhibitor controls to distinguish PPP6C-specific effects from those of other phosphatases

• Substrate validation: Confirm direct dephosphorylation using in vitro phosphatase assays with recombinant PPP6C and phosphorylated substrates

• Mass spectrometry approach: Employ quantitative phosphoproteomics to identify novel phosphorylation sites regulated by PPP6C

Research has identified 288 phosphorylation sites significantly increased after PPP6C depletion, with 57 sites showing decreased phosphorylation, highlighting PPP6C's broad regulatory impact .

How can inducible degradation systems be implemented to study PPP6C function?

Inducible protein degradation offers a powerful approach for studying PPP6C function:

• Targeting strategy: Generate fusion proteins combining PPP6C with degradation tags (e.g., 3xFLAG-sAID-PP6c)

• Genomic integration: Use CRISPR/Cas9 to introduce degradation tags at the endogenous PPP6C locus, maintaining physiological expression levels

• Validation: Confirm system functionality through western blotting, verifying complete band shift from wild-type (35 kDa) to tagged protein (42 kDa)

• Functional assessment: Evaluate whether the tagged PPP6C maintains normal protein interactions through affinity purification and mass spectrometry

• Degradation kinetics: Optimize inducer (e.g., IAA) concentration and treatment duration for rapid, complete protein depletion

Studies have demonstrated the utility of this approach, showing high correlation (R = 0.94) between protein interactions of tagged and untagged PPP6C, suggesting preservation of functional properties despite the modification .

How can phosphoproteomics be leveraged to identify novel PPP6C substrates?

Advanced phosphoproteomic approaches for discovering PPP6C substrates include:

• Acute depletion strategy: Utilize rapid degradation systems (e.g., AID-tagged PPP6C) to minimize compensatory effects and identify direct substrates

• Quantitative analysis: Apply stable isotope labeling (SILAC) or TMT labeling for precise quantification of phosphorylation changes

• Enrichment protocols: Implement sequential enrichment using TiO₂ and IMAC to maximize phosphopeptide coverage

• Bioinformatic filtering: Apply motif analysis to identify phosphosites matching known PPP6C consensus sequences

• Validation pipeline: Confirm direct dephosphorylation through in vitro assays with recombinant PPP6C

This approach has successfully identified phosphorylation sites regulated by PPP6C, revealing 23,278 phosphorylation sites on 5,526 proteins, with 288 sites significantly increased and 57 decreased following PPP6C depletion .

What approaches best reveal PPP6C's role in mitotic regulation?

To investigate PPP6C's mitotic functions:

• Synchronized cell populations: Use mitotic arrest protocols (e.g., nocodazole block) to enrich for mitotic cells and study phase-specific PPP6C activities

• Live-cell imaging: Combine fluorescently tagged PPP6C with time-lapse microscopy to track its dynamic localization during mitosis

• Co-immunoprecipitation: Identify mitosis-specific PPP6C interactors through pulldown experiments from synchronized cells

• Functional assays: Evaluate spindle positioning, chromosome segregation, and mitotic timing in PPP6C-depleted cells

• Substrate analysis: Focus on known mitotic substrates including Aurora A pathway components and Condensin I complex

Research has shown that PPP6C regulates mitotic spindle checkpoint and chromosome cohesion signaling pathways, with gene ontology analyses revealing enrichment in biological processes linked to mitotic cell cycle regulation and chromosome organization .

How can researchers investigate PPP6C's role in immune signaling pathways?

For studying PPP6C in immune regulation:

• Stimulus-specific responses: Compare PPP6C activity and substrate targeting following activation of different immune pathways (e.g., IL-1, cGAS-STING)

• Cellular models: Utilize immune-relevant cell types (macrophages, dendritic cells) alongside non-immune cells to identify context-specific functions

• Infection models: Challenge cells with viral pathogens to assess PPP6C's regulatory role during infection

• Proximity labeling: Apply BioID or APEX2 techniques to identify transient PPP6C interactions during immune stimulation

• Signaling kinetics: Measure activation of downstream transcription factors (IRFs, NF-κB) with temporal resolution after PPP6C manipulation

PPP6C has been demonstrated to regulate innate immunity by acting as a negative regulator of the cGAS-STING pathway through dephosphorylation and inactivation of both CGAS and STING1 . Additionally, it participates in antiviral defense by dephosphorylating RIGI, a critical step for activating RIGI-mediated signaling .

What methods effectively analyze PPP6C holoenzyme composition and dynamics?

To characterize PPP6C holoenzymes:

• Affinity purification: Use tagged PPP6C to isolate intact complexes under physiological conditions

• Blue native PAGE: Separate intact complexes for size estimation and compositional analysis

• Cross-linking mass spectrometry: Identify protein-protein interaction interfaces within the holoenzyme

• Sucrose gradient fractionation: Isolate different PPP6C-containing complexes based on size and density

• Structural studies: Apply cryo-EM to determine the three-dimensional architecture of PPP6C holoenzymes

Affinity purification coupled with mass spectrometry has successfully identified PPP6C's core regulatory subunits (PP6R1-3) and additional interactors including ANKRD28, ANKRD44, ANKRD52, IGBP1, and TIPRL, with high correlation between tagged and untagged PPP6C pulldowns (R = 0.94) .

What factors influence biotin-conjugated antibody performance in multiplexed assays?

When optimizing multiplexed detection with biotin-conjugated PPP6C antibodies:

• Endogenous biotin interference: Pre-block endogenous biotin using avidin/streptavidin treatment in samples with high biotin content

• Signal amplification balance: Titrate streptavidin-conjugate concentration to prevent over-amplification and signal bleeding

• Detection order: In multi-color applications, complete biotin-streptavidin detection before introducing other detection reagents

• Cross-reactivity testing: Validate all antibodies in the multiplex panel to ensure no unexpected cross-reactivity

• Sequential detection protocols: Consider mild stripping between detection steps for complex multiplexing experiments

Biotin-conjugated antibodies targeting amino acids 40-211 of PPP6C have been optimized primarily for ELISA applications and may require additional validation for multiplexed imaging or flow cytometry protocols .

How should researchers interpret contradictory results between different PPP6C antibody clones?

When facing inconsistent results across antibody clones:

• Epitope mapping: Compare the target epitopes of different antibodies (e.g., N-terminal vs. C-terminal regions)

• Post-translational modifications: Consider whether modifications near the epitope might affect antibody binding

• Sample preparation impact: Evaluate whether fixation, denaturation, or buffer conditions differentially affect epitope accessibility

• Cross-reactivity profile: Assess potential cross-reactivity with closely related phosphatases (PP2A, PP4)

• Validation hierarchy: Prioritize data from antibodies with more extensive validation (genetic controls, multiple applications)

Different antibodies target distinct regions of PPP6C, with some recognizing amino acids 40-211 and others targeting C-terminal epitopes , potentially explaining discrepancies in detection patterns or interaction profiles.

What considerations are important when quantifying PPP6C expression or activity levels?

For accurate quantification of PPP6C:

• Loading control selection: Choose loading controls that remain stable under your experimental conditions

• Linear detection range: Establish the linear range of your detection system to ensure quantitative validity

• Signal normalization: Consider dual normalization strategies (total protein and housekeeping genes)

• Activity vs. abundance: Distinguish between PPP6C protein levels and phosphatase activity through activity-specific assays

• Complex formation impact: Account for PPP6C distribution between free and complex-bound states

Western blot analysis using validated PPP6C antibodies can detect both native (35 kDa) and tagged (42 kDa) forms of the protein with high specificity, enabling accurate quantification of expression levels .

What quality control metrics ensure reliable data generation with PPP6C antibodies?

Essential quality control measures include:

• Lot validation: Test each new antibody lot against previous lots using consistent positive controls

• Specificity controls: Include peptide competition controls where feasible

• Reproducibility assessment: Evaluate inter- and intra-assay variability across technical replicates

• Cross-platform validation: Confirm key findings using complementary detection methods

• Recombinant protein standards: Include purified PPP6C protein as a positive control and for generating standard curves

For biotin-conjugated antibodies specifically, additional controls should verify biotin functionality and assess potential interference from endogenous biotin or biotinylated proteins in your experimental system .