ppp1r12a Antibody

What is PPP1R12A and why is it an important research target?

PPP1R12A is a regulatory subunit of protein phosphatase 1 (PP1) that functions as a key regulator of protein phosphatase 1C (PPP1C). In humans, the canonical protein consists of 1030 amino acid residues with a molecular mass of approximately 115.3 kDa . Its importance stems from its role in:

• Myosin phosphatase activity regulation

• Cell morphology and motility control

• Involvement in smooth muscle relaxation

• Recent findings implicate it in YAP-dependent proliferation of cancer cells

The PPP1R12A gene is mapped on chromosome 12q21.2-q21.3, with up to 5 different isoforms reported . Its subcellular localization is primarily cytoplasmic, and it undergoes post-translational modifications, particularly phosphorylation, which significantly affects its function .

What applications are PPP1R12A antibodies most commonly used for?

PPP1R12A antibodies are utilized across multiple experimental applications:

Western Blot is the most widely used application, with over 430 citations describing the use of PPP1R12A antibodies in research . For optimal results, protocols should be adjusted according to the specific antibody manufacturer's recommendations.

How should I select the most appropriate PPP1R12A antibody for my specific research needs?

Selection should be based on:

• Target epitope: Determine if you need antibodies targeting:

  • Total PPP1R12A protein (unphosphorylated)

  • Specific phosphorylation sites (pThr696, pThr853, pSer507)

  • N-terminal vs C-terminal regions

• Species reactivity: Ensure compatibility with your experimental model:

  • Human-specific antibodies

  • Cross-reactive antibodies (human/mouse/rat)

  • Species-specific variations

• Application compatibility: Verify validation for your intended application:

  • Some antibodies perform well in Western blot but poorly in IHC

  • Fixed vs frozen tissue requirements

  • Paraffin-embedded section compatibility

• Clonality consideration:

  • Polyclonal: Better for detection of low-abundance proteins

  • Monoclonal: Higher specificity for particular epitopes

Example: If studying phosphorylation-dependent regulation, select antibodies specifically targeting phospho-sites like pThr696, which is critical for PPP1R12A function regulation .

What are the optimal fixation and antigen retrieval methods for PPP1R12A immunodetection?

For optimal results:

Fixation:

• Paraformaldehyde (PFA) is generally preferred over formalin due to better tissue penetration

• PFA should be freshly prepared before use; long-term stored PFA turns into formalin as molecules congregate

• For cultured cells, 4% PFA for 10-15 minutes at room temperature

Antigen Retrieval for FFPE Tissues:

• Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0, 10mM) for 20 minutes

• Allow sections to cool to room temperature before proceeding with staining

• For phospho-specific antibodies, include phosphatase inhibitors in all buffers

Permeabilization:

• For immunofluorescence of cultured cells, 0.15% Triton X-100 has been successfully used with PPP1R12A antibodies

This protocol has been verified with multiple PPP1R12A antibodies in various human cell lines, including SiHa, U-251 MG, and HeLa cells .

How can I optimize detection of specific PPP1R12A isoforms?

PPP1R12A has up to 5 different reported isoforms , making specific detection challenging. Strategies include:

• Epitope mapping:

  • Select antibodies raised against regions that differ between isoforms

  • Use multiple antibodies targeting different domains to identify variant-specific patterns

• Western blot optimization:

  • Higher percentage gels (8-10%) improve separation of closely sized isoforms

  • Extended running time to enhance band resolution

  • Expected molecular weights vary from canonical 115.3 kDa, with SDS-PAGE observation often at ~150 kDa

• PCR verification:

  • Complement antibody-based detection with isoform-specific PCR

  • Design primers spanning unique exon junctions

• Knockout/knockdown controls:

  • Use siRNA targeting specific isoforms as specificity controls

  • CRISPR/Cas9 modified cell lines can serve as definitive negative controls

Note that the selection of a specific antibody clone can significantly impact isoform detection capabilities. Careful validation with known positive and negative controls is essential.

What strategies are recommended for studying PPP1R12A phosphorylation status?

Phosphorylation of PPP1R12A at specific residues (Thr696, Thr853, Ser507) critically regulates its function. For accurate assessment:

• Phospho-specific antibodies:

  • Use antibodies specifically recognizing pThr696, pThr853, or pSer507

  • Always run paired samples with phosphatase-treated controls

• Sample preparation:

  • Rapid sample processing is crucial to preserve phosphorylation status

  • Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate) in lysis buffers

  • Avoid freeze-thaw cycles that may affect phosphorylation detection

• Signal validation approaches:

  • Lambda phosphatase treatment of parallel samples confirms signal specificity

  • Stimulation with known pathway activators (e.g., Rho-kinase activators for Thr696)

  • Use of kinase inhibitors as negative controls

• Quantification methods:

  • Always normalize phospho-signal to total PPP1R12A levels

  • Use ratio-metric analysis for accurate comparisons between experimental conditions

The phosphorylation status, particularly at Thr696, is critical as it inhibits PPP1R12A activity, with pathogenic variants resulting in non-functional myosin phosphatase .

Why might I observe different molecular weights for PPP1R12A in Western blot analysis?

While the calculated molecular mass of human PPP1R12A is approximately 115.3 kDa , researchers often observe bands at different molecular weights:

To address this variation:

• Validation strategies:

  • Perform siRNA knockdown to confirm specificity of observed bands

  • Use multiple antibodies targeting different epitopes

  • Include positive control lysates with known PPP1R12A expression

• Technical considerations:

  • Optimize gel percentage and running conditions

  • Ensure complete denaturation of samples

  • Consider native vs. reducing conditions impact

The molecular weight discrepancy is not necessarily indicative of non-specific binding, as post-translational modifications significantly affect protein migration patterns.

What are the most effective strategies for reducing background when using PPP1R12A antibodies in immunofluorescence?

Background issues are common challenges in PPP1R12A immunofluorescence studies. Effective mitigation strategies include:

• Blocking optimization:

  • Extended blocking (2 hours at room temperature or overnight at 4°C)

  • Use of species-matched serum (10% goat serum has been effective)

  • Addition of 0.1-0.3% Triton X-100 to blocking buffer for better penetration

• Antibody dilution and incubation:

  • Optimize antibody concentration (typically 2-10 μg/ml for IF)

  • Extended primary antibody incubation (overnight at 4°C)

  • Include 0.05-0.1% Tween-20 in antibody dilution buffer

• Washing protocol enhancement:

  • Increase wash steps (5-6 washes of 5 minutes each)

  • Use PBS-T (PBS with 0.1% Tween-20) for more stringent washing

  • Include one high-salt wash step to reduce non-specific ionic interactions

• Controls implementation:

  • Always include a negative control using isotype-matched non-specific IgG

  • Peptide competition assays to confirm signal specificity

  • Secondary-only controls to assess background from secondary antibody

Documented successful protocol includes paraformaldehyde fixation, 0.15% Triton permeabilization, 10% goat serum blocking, and primary incubation for 1 hour at 10 μg/ml followed by Alexa Fluor 488 secondary antibody at 2 μg/ml .

How can PPP1R12A antibodies be employed to study its role in recycling endosomes and YAP signaling?

Recent research has identified PPP1R12A as a recycling endosomal phosphatase that facilitates YAP activation and cell proliferation . To investigate this emerging role:

• Co-localization studies:

  • Use dual immunofluorescence with PPP1R12A antibodies and markers for recycling endosomes (Rab11, TfR)

  • Super-resolution microscopy for precise localization

• Fractionation experiments:

  • Subcellular fractionation to isolate microsomal fractions

  • Western blot analysis of fractions with PPP1R12A antibodies

  • Compare control cells vs. ATP8A1-depleted cells (ATP8A1 is a PS-flippase that affects PPP1R12A localization)

• Functional analysis:

  • Examine phosphorylated YAP levels after PPP1R12A knockdown

  • Quantify nuclear vs. cytoplasmic YAP distribution

  • Monitor CTGF transcription (a YAP-regulated gene)

• Cancer-relevant models:

  • Focus on triple-negative breast cancer cells (MDA-MB-231) where PPP1R12A is essential for YAP-dependent proliferation

  • Correlate with clinical data from Cancer Genome Atlas

This research direction is particularly relevant as high expression of PPP1R12A correlates with poor prognosis in breast cancer patients, suggesting a potential "ATP8A1-PS-YAP phosphatase" axis in recycling endosomes .

What approaches can be used to study the interaction between PPP1R12A and PP1C using antibodies?

To investigate the functional interaction between PPP1R12A and protein phosphatase type 1 catalytic unit (PP1C):

• Co-immunoprecipitation strategies:

  • Immunoprecipitate with PPP1R12A antibodies and blot for PP1C

  • Reverse co-IP with PP1C antibodies and blot for PPP1R12A

  • Use mild lysis conditions to preserve protein-protein interactions

• Proximity ligation assay (PLA):

  • Enables visualization of protein interactions in situ

  • Requires antibodies from different species for PPP1R12A and PP1C

  • Quantitative assessment of interaction under different conditions

• Phosphatase activity assays:

  • Immunoprecipitate PPP1R12A-PP1C complex with PPP1R12A antibodies

  • Measure phosphatase activity with substrate like myosin light chain

  • Compare activity in control vs. conditions affecting the complex

• Domain mapping:

  • Use antibodies targeting different domains of PPP1R12A

  • Identify regions critical for PP1C binding

  • Correlate with functional outcomes

This approach is significant because PPP1R12A, PP1C, and M20/21 form the trimeric holoenzyme myosin phosphatase, which is a key regulator of cell morphology and motility .

How can PPP1R12A antibodies contribute to understanding pathological conditions?

PPP1R12A has been implicated in several pathological conditions, making antibody-based detection valuable for clinical research:

• Cancer research applications:

  • Tissue microarray analysis of PPP1R12A expression in different cancer types

  • Correlation with clinical outcomes and patient prognosis

  • High expression of PPP1R12A correlates with poor prognosis in breast cancer patients

• Cardiovascular disease studies:

  • PPP1R12A has over 24 publications linking it to cardiovascular diseases

  • Examine phosphorylation status in hypertension models (23 publications)

  • Investigate role in heart failure pathogenesis (4 publications)

• Neurological disorders:

  • PPP1R12A has been linked to nervous system diseases in 5 publications

  • Antibody-based detection in brain tissue samples

  • Correlation with disease progression markers

• Genitourinary and brain malformation syndromes:

  • Pathogenic variants of PPP1R12A gene result in non-functional myosin phosphatase

  • Immunohistochemical analysis of affected tissues

  • Potential diagnostic applications

Researchers should select antibodies validated for the specific tissue types relevant to their pathological focus, as expression patterns may vary significantly between tissues.

What considerations are important when using PPP1R12A antibodies for potential biomarker studies?

When evaluating PPP1R12A as a potential biomarker:

• Antibody selection criteria:

  • High specificity and reproducibility are essential

  • Consider using at least two antibodies targeting different epitopes

  • Select clones validated specifically for diagnostic applications

• Sample preparation standardization:

  • Consistent fixation and processing protocols

  • Standardized antigen retrieval methods

  • Automated staining platforms for reproducibility

• Quantification approaches:

  • Digital pathology for objective scoring

  • Establish clear positive/negative thresholds

  • Consider both intensity and distribution patterns

• Validation requirements:

  • Large cohort validation with appropriate controls

  • Correlation with other established biomarkers

  • Multivariate analysis with clinical parameters

• Special considerations for specific diseases:

  • Breast cancer: Evaluate in context of triple-negative status

  • Cardiovascular disease: Examine phosphorylation at Thr696 and Thr853

  • Neurological disorders: Compare with normal brain expression patterns

This approach is supported by Cancer Genome Atlas data showing correlations between high expression of PPP1R12A, PP1B (catalytic subunit), or ATP8A1 with poor prognosis in breast cancer patients .