PPP1CC Monoclonal Antibody

What is PPP1CC and what are its primary biological functions?

PPP1CC is a serine/threonine protein phosphatase that associates with over 200 regulatory proteins to form highly specific holoenzymes which dephosphorylate hundreds of biological targets . It belongs to the PPP phosphatase family and PP-1 subfamily . PPP1CC plays an important role in dephosphorylating substrates such as the postsynaptic density-associated Ca²⁺/calmodulin dependent protein kinase II . As a protein phosphatase, it's essential for cellular processes including cell division and participates in the regulation of glycogen metabolism, muscle contractility, and protein synthesis. Understanding its function is crucial for researchers studying signaling pathways and their dysregulation in disease states.

How does PPP1CC differ from other protein phosphatase 1 catalytic subunits?

While PPP1CC (gamma isoform) shares functional similarity with other PP1 catalytic subunits like PPP1CB (beta isoform), they have distinct tissue distribution patterns and can form different holoenzyme complexes with regulatory subunits. PPP1CB is involved in regulation of ionic conductances and long-term synaptic plasticity as a component of the PTW/PP1 phosphatase complex, which plays a role in chromatin structure control and cell cycle progression during the transition from mitosis into interphase . In contrast, PPP1CC has been specifically implicated in dephosphorylating postsynaptic density-associated proteins . These differences in substrate specificity and regulatory interactions allow for fine-tuned control of phosphorylation-dependent cellular processes in different tissues and cellular compartments.

What is the molecular weight of PPP1CC and how is this relevant for antibody validation?

The calculated molecular weight of PPP1CC is approximately 38 kDa, though it is commonly observed at around 35 kDa in experimental conditions . This information is critical for antibody validation as researchers must verify that their antibody detects a protein of the correct size in Western blot applications. When validating a PPP1CC monoclonal antibody, the observed band should correspond to this expected molecular weight. Discrepancies between calculated and observed molecular weights may result from post-translational modifications, alternative splicing, or experimental conditions that affect protein migration in SDS-PAGE.

What are the optimal applications for PPP1CC monoclonal antibodies versus polyclonal antibodies?

Monoclonal antibodies against PPP1CC, such as the mouse monoclonal A32527, offer high specificity by detecting endogenous levels of PPP1CC without cross-reacting with related proteins . This makes them ideal for applications requiring precise target recognition. Monoclonals are particularly valuable for:

• Western blot applications requiring high specificity

• Experiments comparing PPP1CC levels across different samples

• Studies where reproducibility between experiments is critical

In contrast, polyclonal antibodies like the sheep polyclonal ab16387 or rabbit polyclonal 55150-1-AP may provide:

• Higher sensitivity due to recognition of multiple epitopes

• Better performance in certain applications like immunoprecipitation

• Greater tolerance to target protein denaturation

The choice depends on your experimental goals: use monoclonals when specificity is paramount and polyclonals when maximum sensitivity is needed.

What is the recommended protocol for using PPP1CC monoclonal antibodies in Western blot applications?

Based on the available data for PPP1CC antibodies, a general Western blot protocol would include:

• Sample preparation: Lyse cells or tissues in an appropriate buffer with protease inhibitors

• Protein quantification and normalization

• SDS-PAGE: Load 20-40 μg of protein per lane

• Transfer to membrane (PVDF or nitrocellulose)

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Dilute PPP1CC monoclonal antibody at 1:500-1:1000 in blocking buffer

• Incubate overnight at 4°C with gentle rocking

• Wash 3-5 times with TBST

• Secondary antibody: Appropriate anti-mouse IgG (for mouse monoclonals) conjugated to HRP at 1:5000-1:10000

• Wash 3-5 times with TBST

• Develop using ECL substrate and image

For optimal results, validate the antibody in your specific experimental system and adjust conditions as needed.

What sample types and species have been validated with commercial PPP1CC monoclonal antibodies?

Commercial PPP1CC monoclonal antibodies have been validated with various sample types and species:

When selecting an antibody for your experiments, ensure it has been validated in species and samples similar to your experimental model. Cross-reactivity with your species of interest is crucial for reliable results. If working with an untested species, sequence homology can provide guidance on potential reactivity, though empirical validation is still recommended.

How can I optimize antibody dilution to reduce background while maintaining signal strength?

Optimizing antibody dilution is crucial for balancing specific signal and background. For PPP1CC monoclonal antibodies:

• Begin with the manufacturer's recommended dilution range (typically 1:500-1:1000 for Western blot)

• Perform a dilution series (e.g., 1:250, 1:500, 1:1000, 1:2000)

• Evaluate signal-to-noise ratio at each dilution

• If background persists despite optimization:

  • Increase blocking time or concentration

  • Add 0.1-0.5% Tween-20 to antibody diluent

  • Consider different blocking agents (BSA vs. milk)

  • Increase washing steps duration and number

  • Pre-absorb the antibody with non-specific proteins

Remember that different applications require different dilutions: immunohistochemistry typically needs higher antibody concentrations (1:20-1:200) compared to Western blot applications.

What are common issues when using PPP1CC antibodies and how can they be resolved?

When working with PPP1CC monoclonal antibodies, researchers may encounter several challenges:

When troubleshooting, change only one variable at a time and maintain detailed records of experimental conditions to identify the source of issues.

How should PPP1CC monoclonal antibodies be stored to maintain activity?

To maintain optimal activity of PPP1CC monoclonal antibodies:

• Store according to manufacturer specifications, typically at -20°C for long-term storage

• For antibodies in glycerol-containing buffers (e.g., 50% glycerol), aliquoting is often unnecessary for -20°C storage

• For antibodies without glycerol, aliquot upon receipt to prevent repeated freeze-thaw cycles

• For working stocks, store at 4°C for up to 2 weeks

• Avoid exposing antibodies to direct light or high temperatures

• Record date of first use and monitor performance over time

Proper storage is critical for maintaining antibody stability and reproducibility across experiments. Some antibodies may show reduced activity over time even with optimal storage, necessitating periodic validation.

How can PPP1CC monoclonal antibodies be employed in studying protein-protein interactions?

PPP1CC monoclonal antibodies can be valuable tools for investigating protein-protein interactions through several approaches:

• Co-immunoprecipitation (Co-IP): Use PPP1CC antibodies to pull down the protein complex, then probe for potential interacting partners. This technique has been validated for PPP1CC antibodies with a recommended amount of 0.5-4.0 μg antibody for 1.0-3.0 mg total protein lysate .

• Proximity Ligation Assay (PLA): Combine PPP1CC monoclonal antibodies with antibodies against suspected interacting partners to visualize and quantify protein interactions in situ.

• Chromatin Immunoprecipitation (ChIP): If investigating PPP1CC's role in transcriptional regulation complexes.

• Immunofluorescence co-localization: Use PPP1CC antibodies in combination with antibodies against potential interacting proteins to assess spatial co-localization.

Given that PPP1CC associates with over 200 regulatory proteins to form specific holoenzymes , these techniques can help elucidate the composition and function of these complexes in different cellular contexts or disease states.

What considerations are important when using PPP1CC antibodies in phosphatase activity assays?

When incorporating PPP1CC monoclonal antibodies in phosphatase activity assays, consider the following:

• Epitope location: Ensure the antibody binds to a region that doesn't interfere with the catalytic site or regulatory protein binding sites. This is particularly important as PPP1CC forms holoenzyme complexes with numerous regulatory proteins .

• Activity neutralization: Determine whether the antibody inhibits, enhances, or has no effect on PPP1CC phosphatase activity before using it in functional studies.

• Complex integrity: Consider whether the antibody disrupts PPP1CC interactions with its regulatory subunits, which could alter substrate specificity and activity.

• Control experiments: Include appropriate controls such as:

  • Irrelevant antibodies of the same isotype

  • Phosphatase inhibitors (e.g., okadaic acid) as positive controls for inhibition

  • Pre-absorption of the antibody with immunizing peptide

• Isoform specificity: Confirm the antibody specifically recognizes PPP1CC and not other PP1 isoforms like PPP1CB , which is critical since different isoforms may have distinct regulatory properties and substrate preferences.

How can PPP1CC antibodies be used to investigate the role of this phosphatase in neuronal function?

PPP1CC plays important roles in neuronal function, particularly in dephosphorylating substrates such as postsynaptic density-associated Ca²⁺/calmodulin dependent protein kinase II . PPP1CC antibodies can be used to investigate these roles through:

• Subcellular localization studies: Use immunofluorescence with PPP1CC monoclonal antibodies in neuronal cultures or brain tissue sections to examine its distribution in dendrites, axons, and synapses. Recommended dilutions for IF range from 1:50-1:500 .

• Activity-dependent changes: Examine how neuronal activity alters PPP1CC localization, expression, or association with regulatory subunits using Western blotting (dilution 1:500-1:1000) after various stimulation paradigms.

• Synaptic plasticity mechanisms: Investigate PPP1CC's role in long-term potentiation (LTP) or depression (LTD) using antibodies to track its recruitment to synapses during plasticity induction.

• Interaction with key neuronal proteins: Use co-immunoprecipitation to identify neuronal-specific binding partners in different brain regions or developmental stages.

• Neurodegenerative disease models: Compare PPP1CC expression, localization, or activity between normal and pathological tissues using immunohistochemistry (recommended dilution 1:20-1:200) .

These approaches can provide insights into how PPP1CC contributes to neuronal development, synaptic function, and neurological disorders.

How should quantitative differences in PPP1CC expression be interpreted across experimental conditions?

When interpreting quantitative differences in PPP1CC expression detected by monoclonal antibodies:

What controls are essential when publishing research using PPP1CC monoclonal antibodies?

When publishing research using PPP1CC monoclonal antibodies, include these essential controls:

• Antibody validation controls:

  • Positive control: Samples known to express PPP1CC (e.g., mouse brain tissue, L02 cells, rat brain tissue)

  • Negative control: Samples lacking PPP1CC expression or knockdown/knockout samples

  • Peptide competition: Pre-incubation of antibody with immunizing peptide to demonstrate specificity

  • Technical replicate: Multiple experiments showing consistent results

• Method-specific controls:

  • For Western blot: Molecular weight markers, loading controls

  • For IHC/ICC: Secondary antibody-only controls, isotype controls

  • For IP: IgG control or unrelated antibody of same isotype

  • For functional assays: Positive and negative controls for the biological process

• Antibody reporting details:

  • Complete antibody information: manufacturer, catalog number, RRID if available

  • Clone designation for monoclonal antibodies

  • Working dilution and incubation conditions

  • Lot number for critical experiments (as lot-to-lot variation can occur)

These controls ensure experimental rigor and reproducibility, which are essential for publication in high-quality journals.

How can researchers distinguish between true PPP1CC signals and artifacts in immunostaining experiments?

Distinguishing genuine PPP1CC signals from artifacts in immunostaining requires rigorous controls and careful analysis:

• Pattern consistency: True PPP1CC staining should show consistent patterns across multiple samples and correspond to known subcellular localization (e.g., cytoplasmic and nuclear distribution) .

• Essential controls:

  • Secondary antibody only: Reveals non-specific binding of secondary antibody

  • Isotype control: Primary antibody of same isotype but irrelevant specificity

  • Peptide competition: Pre-absorption with immunizing peptide should eliminate specific staining

  • Knockdown validation: Reduced signal in samples with PPP1CC knockdown

• Cell type specificity: Compare staining across different cell types with known differences in PPP1CC expression.

• Subcellular localization: PPP1CC localization should be consistent with its known functions in specific cellular compartments.

• Multiple detection methods: Confirm findings using alternative antibodies or detection techniques.

• Fixation artifacts: Different fixation methods can affect epitope accessibility; compare multiple preparation methods when possible.

By systematically implementing these approaches, researchers can confidently distinguish true PPP1CC signals from technical artifacts, enhancing the reliability of immunostaining results.