PPP1R12C Antibody

What is PPP1R12C and what cellular functions does it regulate?

PPP1R12C (protein phosphatase 1, regulatory inhibitor subunit 12C) functions as a regulatory subunit of myosin phosphatase. It specifically regulates the catalytic activity of protein phosphatase 1 delta and plays a crucial role in the assembly of the actin cytoskeleton . The protein has a calculated molecular weight of 85 kDa (782 amino acids) but is typically observed at 90-100 kDa in experimental conditions . PPP1R12C forms a holoenzyme complex with PP1 catalytic subunit (PP1c), directing its activity toward specific substrates including myosin light chain 2 (MLC2a) in cardiac tissue. This regulatory function is critical for controlling cellular contractility mechanisms, particularly in cardiac muscle cells where it modulates the phosphorylation state of contractile proteins.

What applications are PPP1R12C antibodies validated for in research settings?

PPP1R12C antibodies have been validated for multiple research applications with varying recommended dilutions and protocols:

Researchers should note that optimal dilutions may be sample-dependent, and it is recommended to titrate the antibody in each specific testing system to obtain optimal results . The antibodies show reactivity with human, mouse, and rat samples, making them versatile for comparative studies across species .

How should PPP1R12C antibodies be stored and handled to maintain reactivity?

For optimal performance, PPP1R12C antibodies should be stored at -20°C, where they remain stable for one year after shipment . The storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Importantly, aliquoting is generally unnecessary for -20°C storage, simplifying laboratory protocols . Some preparations, particularly those in 20μl sizes, contain 0.1% BSA which helps stabilize the antibody . When working with these antibodies, researchers should avoid repeated freeze-thaw cycles and exposure to strong light or contaminating enzymes that could degrade protein structure.

How can PPP1R12C antibodies be used to investigate atrial fibrillation mechanisms?

Recent research has established a critical link between PPP1R12C and atrial fibrillation (AF), with PPP1R12C overexpression promoting atrial hypocontractility in AF patients . When designing experiments to investigate this relationship, researchers should consider:

• Comparative expression analysis: Western blot techniques using PPP1R12C antibodies revealed that AF patients exhibit a 2-fold increase in PPP1R12C expression compared to sinus rhythm controls (P=2.0×10⁻²; n=12 in each group) .

• Phosphorylation state assessment: PPP1R12C antibodies can be used in conjunction with phospho-specific antibodies to evaluate MLC2a phosphorylation, which is reduced by >40% in AF patients (P=1.4×10⁻⁶) .

• Interaction studies: Co-immunoprecipitation experiments using PPP1R12C antibodies demonstrated increased PPP1R12C-PP1c binding (P=2.9×10⁻²) and PPP1R12C-MLC2a binding (P=6.7×10⁻³) in AF patients .

The methodological approach should include multiple controls and standardization of tissue collection procedures when comparing pathological samples to ensure reproducible results.

What are the optimal experimental designs for studying PPP1R12C interactions with binding partners?

When investigating PPP1R12C protein-protein interactions, researchers should implement the following methodological approaches:

• Co-immunoprecipitation (Co-IP): Use PPP1R12C antibodies to pull down the protein complex, followed by Western blotting for suspected binding partners such as PP1c and MLC2a . This technique has successfully demonstrated increased binding between PPP1R12C and both PP1c and MLC2a in atrial fibrillation conditions.

• Proximity ligation assays: For detecting in situ protein interactions with spatial resolution.

• Pharmacological manipulation: Studies utilizing the MRCK inhibitor BDP5290, which inhibits T560-PPP1R12C phosphorylation, have demonstrated that phosphorylation state affects PPP1R12C binding affinity to both PP1c and MLC2a .

• Genetic models: Cardiac-specific lentiviral PPP1R12C overexpression in mice provides a valuable tool for examining the functional consequences of altered PPP1R12C expression on atrial remodeling and contractility .

When designing these experiments, it's critical to include appropriate negative controls and validate antibody specificity through multiple approaches to ensure result reliability.

How can researchers distinguish between PPP1R12C and other PP1 regulatory subunits in experimental setups?

Distinguishing between different PP1 regulatory subunits requires careful experimental design:

• Antibody selection: Choose PPP1R12C antibodies raised against unique epitopes that don't cross-react with other PP1 regulatory subunits. The immunogen information indicates that antibodies such as 25157-1-AP target specific PPP1R12C fusion protein regions (Ag18559) .

• Validation protocols:

  • Perform Western blots on tissues with differential expression of PP1 regulatory subunits

  • Include positive control samples known to express PPP1R12C (e.g., mouse colon tissue, HEK-293T cells, heart tissues)

  • Use siRNA knockdown of PPP1R12C to confirm antibody specificity

  • Consider mass spectrometry validation of immunoprecipitated proteins

• Expression pattern analysis: PPP1R12C demonstrates tissue-specific expression patterns that can be leveraged to distinguish it from other regulatory subunits, with particular attention to cardiac tissue where its role in atrial function has been established .

What troubleshooting approaches are recommended when PPP1R12C antibodies show unexpected results in Western blot applications?

When encountering unexpected results with PPP1R12C antibodies in Western blotting:

• Molecular weight verification: PPP1R12C has a calculated molecular weight of 85 kDa but is typically observed at 90-100 kDa . Discrepancies may indicate post-translational modifications, isoform variation, or degradation.

• Sample preparation optimization:

  • Ensure complete protein denaturation and reduction

  • Verify protein extraction buffer compatibility with phosphorylated protein preservation

  • Consider phosphatase inhibitors when studying PPP1R12C phosphorylation states

• Antibody dilution optimization: The recommended dilution range for Western blotting is 1:5000-1:50000 , but sample-dependent optimization is crucial for optimal signal-to-noise ratio.

• Control experiments:

  • Include positive control tissues known to express PPP1R12C (mouse colon, HEK-293T cells, heart tissues)

  • Consider using recombinant PPP1R12C as a reference standard

  • Perform antibody validation using PPP1R12C overexpression or knockdown models

• Protocol modifications:

  • Adjust blocking conditions to reduce background

  • Optimize incubation times and temperatures

  • Consider alternative membrane types if protein transfer efficiency is suboptimal

How should researchers design experiments to investigate PPP1R12C's role in the dephosphorylation of myosin light chain in cardiac tissue?

Investigating PPP1R12C's role in MLC2a dephosphorylation requires a multifaceted experimental approach:

• Tissue-specific analysis: Human atrial appendage tissues from AF patients versus sinus rhythm controls have demonstrated significant differences in PPP1R12C expression and MLC2a phosphorylation .

• Phosphorylation assessment protocols:

  • Use phospho-specific antibodies for MLC2a alongside PPP1R12C antibodies

  • Employ Phos-tag gels for enhanced separation of phosphorylated versus non-phosphorylated proteins

  • Consider quantitative mass spectrometry approaches for unbiased phosphorylation site identification

• Functional correlation studies:

  • In vitro cell shortening assays to correlate PPP1R12C expression with contractile function

  • Echocardiography in animal models to assess atrial size and function (Lenti-PPP1R12C mice showed 150% increase in left atrial size, P=5.0×10⁻⁶)

  • Electrophysiology studies to evaluate AF inducibility (significantly higher in Lenti-PPP1R12C mice, P=1.8×10⁻² and 4.1×10⁻²)

• Pharmacological manipulation:

  • MRCK inhibitor BDP5290 affects T560-PPP1R12C phosphorylation, providing a tool to modulate PPP1R12C activity and study downstream effects on MLC2a phosphorylation

What are the key considerations for validating new PPP1R12C antibodies for research applications?

Comprehensive validation of new PPP1R12C antibodies should include:

• Specificity assessment:

  • Western blotting against tissues known to express PPP1R12C (colon, heart) versus negative controls

  • Immunoprecipitation followed by mass spectrometry identification

  • Testing in PPP1R12C knockout or knockdown systems

  • Epitope mapping to confirm target sequence specificity

• Application-specific validation:

  • For Western blotting: Verify appropriate molecular weight (90-100 kDa observed) and single band specificity

  • For immunoprecipitation: Confirm pull-down efficiency and specificity

  • For immunofluorescence: Validate subcellular localization pattern consistent with PPP1R12C biology

  • For immunohistochemistry: Compare staining patterns with known expression data

• Cross-species reactivity verification:

  • Test against human, mouse, and rat samples as PPP1R12C antibodies like 25157-1-AP show reactivity with these species

  • Align target epitope sequences across species to predict potential cross-reactivity

• Reproducibility testing:

  • Perform inter-laboratory validation when possible

  • Test multiple antibody lots for consistent performance

  • Validate across different sample types and experimental conditions

How should researchers interpret PPP1R12C expression changes in the context of cardiovascular disease progression?

Research indicates that PPP1R12C upregulation plays a significant role in cardiac pathophysiology, particularly in atrial fibrillation:

• Expression level interpretation:

  • A 2-fold increase in PPP1R12C expression has been observed in AF patients compared to controls (P=2.0×10⁻²)

  • Changes should be interpreted in relation to functional outcomes, such as the 40% reduction in MLC2a phosphorylation (P=1.4×10⁻⁶)

• Correlation with disease severity:

  • Increased PPP1R12C binding to PP1c and MLC2a correlates with atrial hypocontractility

  • Animal models with PPP1R12C overexpression demonstrate increased susceptibility to pacing-induced AF

• Causal relationship assessment:

  • Lentiviral PPP1R12C overexpression in mice leads to 150% increase in left atrial size (P=5.0×10⁻⁶)

  • These models show reduced atrial strain and ejection fraction, suggesting a direct causal relationship between PPP1R12C expression and atrial dysfunction

• Therapeutic target potential:

  • The established relationship between PPP1R12C, MLC2a dephosphorylation, and atrial dysfunction suggests PPP1R12C as a potential therapeutic target

  • Researchers should design experiments to evaluate whether inhibiting PPP1R12C activity or expression can reverse atrial hypocontractility

What experimental approaches can distinguish between correlation and causation when studying PPP1R12C in atrial fibrillation?

Distinguishing between correlation and causation requires rigorous experimental design:

• Genetic manipulation models:

  • Cardiac-specific lentiviral PPP1R12C overexpression in mice has established causality by demonstrating that increased PPP1R12C leads to atrial enlargement, reduced contractility, and increased AF susceptibility

  • Consider conditional knockout models to determine if PPP1R12C reduction can prevent or reverse AF-associated changes

• Temporal relationship studies:

  • Time-course experiments to determine whether PPP1R12C upregulation precedes or follows the onset of atrial dysfunction

  • Longitudinal studies in animal models to track progression from PPP1R12C overexpression to functional changes

• Dose-response relationships:

  • Titrated expression systems to determine if the degree of PPP1R12C upregulation correlates with the severity of atrial dysfunction

  • Quantitative analysis of PPP1R12C-PP1c binding and subsequent MLC2a dephosphorylation

• Intervention studies:

  • Pharmacological interventions targeting PPP1R12C activity, such as MRCK inhibitor BDP5290 which affects T560-PPP1R12C phosphorylation

  • Rescue experiments to determine if normalizing PPP1R12C levels or activity can reverse established atrial dysfunction

How can researchers optimize co-immunoprecipitation protocols to study native PPP1R12C complexes in cardiac tissue?

Optimizing co-immunoprecipitation for PPP1R12C complexes requires special consideration:

• Lysis buffer composition:

  • Use mild detergents (0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions

  • Include phosphatase inhibitors to maintain phosphorylation states, particularly important for studying T560-PPP1R12C phosphorylation

  • Consider including protease inhibitors to prevent degradation during sample processing

• Antibody selection and validation:

  • Choose PPP1R12C antibodies that recognize native protein conformations

  • Validate that the antibody's epitope is not involved in or blocked by protein-protein interactions

  • Consider using multiple antibodies targeting different epitopes to confirm results

• Technical modifications:

  • Cross-linking strategies can stabilize transient interactions

  • Sequential immunoprecipitation can help identify multi-protein complexes

  • Native gel electrophoresis followed by Western blotting can preserve and identify intact complexes

• Control experiments:

  • Include IgG controls to account for non-specific binding

  • Perform reverse co-immunoprecipitation (precipitating with antibodies against suspected binding partners)

  • Use tissues from disease models (AF) and controls to compare complex formation under different physiological conditions

What approaches should researchers use to investigate post-translational modifications of PPP1R12C that affect its function?

Investigating PPP1R12C post-translational modifications requires specialized techniques:

• Phosphorylation analysis:

  • Phospho-specific antibodies targeting known sites like T560

  • Phos-tag SDS-PAGE for mobility shift detection

  • Mass spectrometry-based phosphoproteomics for unbiased site identification

  • In vitro kinase assays to identify enzymes responsible for PPP1R12C phosphorylation

• Functional correlation studies:

  • Site-directed mutagenesis of key phosphorylation sites (e.g., T560 to alanine to prevent phosphorylation)

  • Phosphomimetic mutations (e.g., T560 to glutamic acid)

  • MRCK inhibitor BDP5290 to modulate T560-PPP1R12C phosphorylation and observe effects on binding partners

• Temporal dynamics:

  • Pulse-chase experiments to determine modification turnover rates

  • Synchronized cell systems to examine cell-cycle dependent modifications

  • Acute stimulation protocols to capture rapid changes in modification status

• Localization impact:

  • Combine phospho-specific antibodies with subcellular fractionation or immunofluorescence

  • Determine if modifications alter PPP1R12C subcellular distribution or complex formation