PPP3CC Antibody

What is PPP3CC and what cellular functions does it perform?

PPP3CC, also known as CALNA3 or CNA3, is a calcium-dependent, calmodulin-stimulated protein phosphatase belonging to the PPP phosphatase family and PP-2B subfamily. It functions as the gamma catalytic subunit of calcineurin. PPP3CC plays essential roles in cellular signaling by:

• Dephosphorylating and inactivating transcription factor ELK1

• Dephosphorylating DARPP32

• Participating in calmodulin activation of calcineurin

The protein has a calculated molecular weight of approximately 58 kDa and is encoded by a gene located at chromosome 8p21.3 in humans .

Which tissue types express PPP3CC and how can this inform antibody validation?

PPP3CC shows differential expression across tissues, which is critical for antibody validation:

When validating a PPP3CC antibody, researchers should include positive controls from tissues known to express the protein (particularly testis and brain tissues) and negative controls from tissues with minimal expression .

What are the primary applications for PPP3CC antibodies?

PPP3CC antibodies have been validated for multiple research applications:

Most commercially available antibodies are polyclonal and raised in rabbit hosts, though some monoclonal options are available .

How should researchers optimize Western blotting protocols for PPP3CC detection?

For optimal Western blot detection of PPP3CC:

• Sample preparation: Use approximately 30 μg of total protein as suggested in published research

• Control selection: Include both positive controls (testis tissue) and negative controls

• Antibody dilution: Start with 1:500-1:1000 dilution and optimize based on signal-to-noise ratio

• Detection system: Both chemiluminescence and fluorescence-based systems work effectively

• Molecular weight confirmation: Verify that the detected band appears at the expected 57-58 kDa

• Loading control: GAPDH has been successfully used as an internal reference in PPP3CC studies

For researchers investigating expression differences in pathological conditions, quantitative Western blot with appropriate normalization is recommended, as significant differences in PPP3CC protein levels have been observed between normal and disease states .

What is the relationship between PPP3CC expression and sperm motility, and how can antibodies help investigate this connection?

Research has established a significant correlation between PPP3CC expression and sperm motility:

• Expression correlation: PPP3CC protein levels are significantly lower in asthenozoospermia (AZS) compared to normal sperm samples

• Functional evidence: Calcineurin inhibitors (like cyclosporine A and tacrolimus) reduce sperm motility, mimicking the AZS phenotype

• Quantitative relationship: PPP3CC protein content positively correlates with sperm progressive motility (PR)

• mRNA vs protein discrepancy: Interestingly, while PPP3CC mRNA levels differ between AZS and normal sperm, there's no significant correlation between mRNA levels and PR or between mRNA and protein levels in the same samples

For researchers investigating male fertility issues, PPP3CC antibodies can be used to:

• Quantify protein expression via Western blotting with densitometry analysis

• Localize PPP3CC within sperm cells via immunofluorescence

• Correlate expression with clinical parameters such as progressive motility

How can researchers investigate PPP3CC's role in cancer biology using specific antibodies?

PPP3CC has emerging roles in cancer biology that can be investigated using specific approaches:

• Expression analysis: PPP3CC has been identified as a potential tumor suppressor in epithelial ovarian cancer (EOC)

• Regulatory mechanisms: miR-200c-3p targets and inhibits PPP3CC in EOC, with an inverse correlation between miR-200c-3p and PPP3CC expression

• Functional effects: PPP3CC knockdown in UWB 1.289 + BRCA1 cell line induces proliferation and metastasis, suggesting tumor suppressor properties

• Pathway interactions: PPP3CC affects apoptosis and AKT signaling pathways in cancer cells

Methodological approaches for cancer research:

• Use PPP3CC antibodies for comparative expression analysis between normal and tumor tissues

• Combine with phospho-specific antibodies to examine downstream signaling effects

• For knockout/knockdown validation experiments, confirm PPP3CC reduction using validated antibodies

• In patient samples, correlate PPP3CC expression with clinical outcomes and disease progression

What methodological considerations are important for immunohistochemical detection of PPP3CC?

For successful IHC detection of PPP3CC:

• Antigen retrieval: Most protocols recommend TE buffer pH 9.0, though citrate buffer pH 6.0 can be used as an alternative

• Antibody dilution: Start with 1:50-1:100 for formalin-fixed paraffin-embedded (FFPE) tissues and optimize as needed

• Positive control tissues: Human stomach tissue has been validated as a reliable positive control

• Specificity validation: When possible, include known negative tissues or validate using peptide competition assays

• Detection systems: Both chromogenic and fluorescent detection methods are compatible

• Cross-reactivity assessment: Several PPP3CC antibodies show cross-reactivity with human, mouse, and rat samples

For specialized applications like detecting PPP3CC in reproductive tissues, researchers may need to adjust fixation times and antigen retrieval conditions for optimal results.

How do different epitopes affect antibody selection for PPP3CC research?

PPP3CC antibodies targeting different epitopes provide various advantages depending on research objectives:

For researchers investigating protein-protein interactions or studying post-translational modifications, antibody selection should consider:

• Whether the epitope overlaps with functional domains

• If the epitope contains known modification sites

• Potential masking of the epitope during complex formation

How can researchers address issues with PPP3CC antibody specificity and sensitivity?

When encountering specificity or sensitivity issues:

• Verify sample preparation: Ensure proper protein extraction, especially from tissues with high protease activity

• Blocking optimization: Test different blocking agents (BSA vs. milk) to reduce background

• Antibody validation: Confirm antibody specificity using:

  • Positive control tissues (testis, brain)

  • Knockout/knockdown validation

  • Peptide competition assays

• Cross-reactivity: Be aware that some PPP3CC antibodies may cross-react with other calcineurin subunits

• Signal enhancement: For low-abundance detection, consider using signal amplification methods or more sensitive detection systems

What are the most effective approaches for studying PPP3CC in relation to calcineurin activity?

To investigate PPP3CC's role in calcineurin function:

• Activity correlation: Combine PPP3CC antibody detection with calcineurin activity assays to correlate expression and function

• Complex formation: Use co-immunoprecipitation with PPP3CC antibodies to pull down interacting partners

• Inhibitor studies: Compare PPP3CC expression/localization in the presence of calcineurin inhibitors like cyclosporine A or tacrolimus

• Calcium dependence: Examine PPP3CC localization and complex formation under varying calcium conditions

• Regulatory subunit interaction: Investigate the relationship between PPP3CC and PPP3R2 (regulatory subunit), which have shown different patterns in certain pathologies

What considerations are important when using PPP3CC antibodies for reproductive biology research?

For reproductive biology applications:

• Tissue preparation: Testicular tissue requires careful fixation and antigen retrieval optimization

• Expression correlation: When studying infertility, correlate PPP3CC expression with clinical parameters such as sperm motility

• Comparative analysis: Consider analyzing both PPP3CC and PPP3R2 expression, as research has shown PPP3CC, but not PPP3R2, correlates with asthenozoospermia

• Functional studies: Combine antibody-based detection with calcineurin inhibitor studies to examine functional effects on sperm

• Protein-mRNA correlation: Be aware that PPP3CC protein and mRNA levels may not correlate in sperm samples, necessitating both protein and transcript analysis

This discrepancy between mRNA and protein levels makes antibody-based detection particularly important in reproductive research.

How should experimental design address the confounding factors in PPP3CC expression analysis?

When designing experiments to analyze PPP3CC expression:

• Tissue heterogeneity: Account for cell-type specific expression, particularly in tissues like brain or testis

• Sample preparation variability: Standardize protein extraction methods to ensure consistent detection

• Expression regulation: Consider that PPP3CC can be regulated at multiple levels (transcriptional, post-transcriptional, post-translational)

• miRNA regulation: In cancer studies, account for miRNA regulation (such as miR-200c-3p) that can affect PPP3CC levels

• Isoform detection: Be aware of potential splice variants and design experiments to distinguish between them

• Cross-reactivity: Validate antibody specificity against other calcineurin catalytic subunits (PPP3CA, PPP3CB)

What are the emerging applications of PPP3CC antibodies in neuroscience research?

For neuroscience applications:

• Brain expression: PPP3CC is expressed in brain tissue and may have region-specific functions

• Calcineurin signaling: PPP3CC participates in calcium-dependent signaling pathways critical for neuronal function

• Substrate interactions: Investigate PPP3CC's relationship with neuronal substrates such as DARPP32

• Localization studies: Use immunofluorescence to determine subcellular localization in neurons

• Disease associations: Explore potential associations with neurological disorders

Researchers should:

• Use brain tissue as positive controls for antibody validation

• Consider region-specific expression patterns when designing experiments

• Combine with neuronal markers for co-localization studies

How can researchers accurately quantify changes in PPP3CC expression using antibody-based methods?

For precise quantification of PPP3CC expression changes:

• Quantitative Western blotting: Use standard curves with recombinant protein for absolute quantification

• Digital imaging: Apply digital image analysis for densitometry with appropriate controls

• Normalization strategy: Test multiple housekeeping proteins (GAPDH has been validated)

• Statistical analysis: Apply appropriate statistical tests for comparing expression between groups

• Sample size calculation: Base sample size on expected effect size and variability in PPP3CC expression

• Reproducibility: Include biological and technical replicates to ensure reliable quantification

In studies comparing normal and pathological states, researchers should be particularly careful with normalization and statistical analysis to detect meaningful differences in PPP3CC expression.

What novel approaches are being developed for studying PPP3CC function beyond traditional antibody applications?

Emerging methodologies for PPP3CC research include:

• CRISPR/Cas9 modifications: Gene editing to create tagged versions of endogenous PPP3CC for live-cell imaging

• Proximity labeling: BioID or APEX2 fusions to identify novel interaction partners

• Single-cell analysis: Combining antibody-based detection with single-cell transcriptomics

• PTM-specific antibodies: Development of antibodies specific to post-translationally modified forms of PPP3CC

• Super-resolution microscopy: Nanoscale localization of PPP3CC within cellular compartments

• Interactome mapping: Using antibodies for immunoprecipitation followed by mass spectrometry