PPP1R9A Antibody

What is PPP1R9A and why is it important in neurological research?

PPP1R9A (Protein Phosphatase 1 Regulatory Subunit 9A), also known as Neurabin-I, is a brain-specific protein that contains an F-actin binding domain, a PDZ domain, a transmembrane-protein-interacting domain, and a coiled-coil region. This multidomain protein binds to F-actin, protein phosphatase 1, TGN38, and p70 S6 kinase . PPP1R9A is highly concentrated in neuronal synapses and is enriched in the lamellipodia of growth cones during neuronal development. It functions as a bridging protein by targeting other proteins to the synapse and linking membrane proteins to the actin cytoskeleton . Its role in neurite formation and protein phosphatase inhibition makes it a critical target for neurological research, particularly in studies of synaptic plasticity and neuronal development.

What types of PPP1R9A antibodies are available for research applications?

Several types of PPP1R9A antibodies are available for research:

Researchers should select antibodies based on their specific application needs and the species being studied, with rabbit polyclonals being the most widely available option with broad application potential .

What is the difference between PPP1R9A, Neurabin-I, and NRB1 in antibody product listings?

These are alternative names for the same protein. PPP1R9A is the official gene symbol, while Neurabin-I (or Neurabin-1) and NRB1 are commonly used aliases . When searching for antibodies, it's important to consider all these aliases as different manufacturers may list their products under various names:

• PPP1R9A: Protein Phosphatase 1 Regulatory Subunit 9A (official name)

• Neurabin-I/Neurabin-1: Neural tissue-specific F-actin binding protein I

• NRB1: Another common alias

• KIAA1222: Another identifier sometimes used

When ordering antibodies, confirm the target protein's full identity by checking UniProt IDs (Q9ULJ8 for human PPP1R9A) or GenBank accession numbers (BC150636) .

What are the optimal dilutions for using PPP1R9A antibodies in different applications?

Based on manufacturer recommendations, optimal dilutions vary by application:

It is strongly recommended to titrate antibodies in each specific testing system to obtain optimal results, as dilution requirements can be sample-dependent . Validation data galleries from manufacturers can provide guidance for specific cell lines and tissues.

What cell lines have been validated for detecting endogenous PPP1R9A expression?

Several cell lines have been validated for PPP1R9A detection:

While PPP1R9A is described as brain-specific in some literature , commercial antibodies have successfully detected it in non-neuronal cell lines, suggesting either low-level expression in multiple tissues or potential cross-reactivity that should be carefully validated in experimental contexts.

How should PPP1R9A antibodies be stored to maintain optimal activity?

Proper storage is critical for antibody performance:

• Long-term storage: Store at -20°C, where antibodies remain stable for approximately one year after shipment

• Short-term storage: 4°C is suitable for 1-2 weeks

• Very long-term storage: Some manufacturers recommend -80°C for extended periods

• Buffer composition: Typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Aliquoting: While some manufacturers state aliquoting is unnecessary for -20°C storage , dividing antibodies into single-use aliquots is generally recommended to prevent repeated freeze-thaw cycles

Most manufacturers indicate that their PPP1R9A antibodies remain stable for one year after shipment when stored properly. Note that small volume sizes (20μl) may contain 0.1% BSA as a stabilizer .

How can PPP1R9A antibodies be utilized to study its role in neurological disorders?

PPP1R9A has emerging significance in neurological research, particularly regarding PPP1R9A-related syndrome:

• Genetic screening validation: Antibodies can confirm protein expression levels in patient-derived samples carrying PPP1R9A variants .

• Functional studies: Immunofluorescence can localize PPP1R9A in neuronal cultures to study its role in:

  • Neurite formation

  • Synaptic development

  • Synaptic plasticity alterations in disease models

• Protein-protein interaction studies: Co-immunoprecipitation with PPP1R9A antibodies can identify interaction partners in normal vs. pathological conditions, particularly its association with protein phosphatase 1 and actin cytoskeleton .

• Therapeutic screening: Western blot analysis can assess changes in PPP1R9A levels in response to potential therapeutic compounds targeting PPP1R9A-related pathways.

Since PPP1R9A-related syndrome is newly identified with limited research, rigorous validation of antibody specificity is essential when studying patient samples .

What is the significance of the discrepancy between calculated (123 kDa) and observed (180 kDa) molecular weights of PPP1R9A?

The significant difference between calculated (123 kDa) and observed (180 kDa) molecular weights of PPP1R9A warrants careful consideration:

• Post-translational modifications: The higher observed molecular weight likely reflects extensive post-translational modifications such as phosphorylation, glycosylation, or SUMOylation.

• Isoform variation: At least 5 isoforms of human NRB1/PPP1R9A have been identified , which may run at different molecular weights.

• Experimental interpretation: When performing Western blots:

  • Always include positive controls from validated cells (e.g., PC-3 cells)

  • Be aware that antibodies may detect multiple bands representing different isoforms or modification states

  • Consider using phosphatase treatments to determine if phosphorylation contributes to the shift

• Functional implications: The substantial size difference suggests extensive regulation that may be physiologically relevant to PPP1R9A's scaffolding functions in neurons.

Researchers should document the exact molecular weight observed in their experimental system and consider investigating the nature of modifications if relevant to their research question.

How does PPP1R9A expression in cancer cells relate to its neuronal functions, and what antibody-based approaches can investigate this connection?

Recent research has implicated PPP1R9A in cancer biology:

• Expression analysis: PPP1R9A has been found upregulated in hepatocellular carcinoma (HCC), correlating with unfavorable prognosis . Immunohistochemistry using validated PPP1R9A antibodies can assess expression levels across cancer types and stages.

• Mechanistic roles: PPP1R9A is involved in regulating cell adhesion, which is essential for cancer cell invasion and metastasis . Specific antibody applications include:

  • Immunofluorescence to study localization changes during epithelial-mesenchymal transition

  • Proximity ligation assays to detect altered protein-protein interactions in cancer cells

  • Chromatin immunoprecipitation to investigate potential transcriptional regulation roles

• Pathway analysis: Gene Set Enrichment Analysis has linked PPP1R9A to pathways including MAPK, AMPK, and PI3K-Akt signaling . Co-immunostaining with pathway components can validate these connections.

• Potential as biomarker: The significant expression of PPP1R9A in HCC suggests potential as a diagnostic/prognostic marker , which could be developed into antibody-based diagnostic assays.

When investigating PPP1R9A in cancer contexts, researchers should validate antibody specificity in relevant cancer cell lines, as expression patterns may differ significantly from neuronal models.

What are common issues when using PPP1R9A antibodies in Western blotting, and how can they be addressed?

When using PPP1R9A antibodies for Western blotting, researchers may encounter several challenges:

Follow manufacturer's specific protocols when available. For example, Proteintech offers specific WB protocols for their PPP1R9A antibody (18801-1-AP) .

How can PPP1R9A antibody specificity be validated in experimental systems?

Thorough validation of PPP1R9A antibodies is essential:

• Positive controls: Use cell lines with confirmed PPP1R9A expression (PC-3 for WB; HepG2 or HeLa for IF/ICC)

• Negative controls:

  • Genetic: siRNA knockdown, CRISPR knockout

  • Technical: Omission of primary antibody, isotype controls

  • Pre-absorption: Pre-incubate antibody with immunizing peptide if available

• Orthogonal validation:

  • Compare results from multiple antibodies targeting different epitopes

  • Correlate antibody staining with mRNA expression data

  • Confirm subcellular localization matches reported patterns (enriched in synapses and lamellipodia of growth cones)

• Application-specific validation:

  • For IHC: Compare staining patterns with in situ hybridization results

  • For IP: Confirm pulled-down protein by mass spectrometry

  • For IF: Co-localization with known interacting partners like F-actin

Documentation of validation experiments enhances reproducibility and reliability of research findings.

What considerations are important when using PPP1R9A antibodies for studying post-translational modifications?

Studying post-translational modifications (PTMs) of PPP1R9A requires specific methodological considerations:

• Phosphorylation analysis:

  • Use phosphatase inhibitors in lysate preparation

  • Consider phospho-specific antibodies if available

  • Perform lambda phosphatase treatment as control

  • The large difference between observed (180 kDa) and calculated (123 kDa) MW suggests extensive phosphorylation

• Sample preparation:

  • Use appropriate lysis buffers that preserve PTMs

  • Avoid excessive sample heating

  • Process samples quickly to prevent modification loss

• Co-immunoprecipitation studies:

  • When studying PPP1R9A interactions with protein phosphatase 1, preserve physiological conditions

  • Consider crosslinking approaches for transient interactions

• Advanced techniques:

  • Combine with mass spectrometry to identify specific modified residues

  • Use proximity ligation assays to detect specific PPP1R9A interactions in situ

  • Consider FRET-based approaches for dynamic interaction studies

When publishing results, clearly document all sample preparation steps and modification-preserving measures used in the experimental workflow.

How are PPP1R9A antibodies being used in studies of PPP1R9A-related syndrome?

PPP1R9A-related syndrome is an emerging genetic condition caused by variants in the PPP1R9A gene. Antibody-based approaches for studying this syndrome include:

• Protein expression analysis:

  • Western blotting to quantify PPP1R9A levels in patient-derived samples

  • Immunofluorescence to assess subcellular localization changes caused by mutations

• Functional impact assessment:

  • Immunoprecipitation to determine if mutations alter protein-protein interactions

  • Co-localization studies with synaptic markers in neuronal cultures from patient iPSCs

• Diagnostic development:

  • As of 2024, PPP1R9A-related syndrome patients are still being identified

  • Antibody-based screening could help identify additional cases

• Methodological challenges:

  • Limited patient samples necessitate careful experimental design

  • Control samples should be age and background-matched

  • Variant-specific effects may require personalized approaches

Since PPP1R9A-related syndrome research is still nascent, antibody-based studies can make significant contributions to understanding disease mechanisms .

What techniques combine PPP1R9A antibodies with advanced imaging to study neuronal function?

Advanced imaging techniques using PPP1R9A antibodies offer powerful insights into neuronal function:

• Super-resolution microscopy:

  • STORM/PALM imaging reveals PPP1R9A nanoscale organization at synapses

  • Correlate with actin cytoskeleton using multi-color super-resolution

  • Protocol optimization: use primary antibodies at 1:10-1:100 dilution , followed by appropriate fluorophore-conjugated secondary antibodies

• Live-cell imaging approaches:

  • Combine with fluorescently tagged PPP1R9A for dynamic studies

  • FRAP (Fluorescence Recovery After Photobleaching) to study mobility

  • Use neuronal cultures from validated cell lines where PPP1R9A expression is confirmed

• Multi-modal correlative microscopy:

  • Combine immunofluorescence with electron microscopy

  • Precisely localize PPP1R9A at ultrastructural level in synapses

• Tissue clearing and 3D imaging:

  • Whole-brain immunostaining followed by clearing techniques (CLARITY, iDISCO)

  • Map PPP1R9A distribution across neural circuits

These techniques require careful optimization of antibody penetration, concentration, and specificity validation to avoid artifacts in complex neuronal preparations.

How should researchers quantify and analyze PPP1R9A expression levels across different experimental conditions?

Robust quantification of PPP1R9A expression requires methodological rigor:

• Western blot quantification:

  • Use appropriate loading controls (β-actin, GAPDH)

  • Note that observed molecular weight (180 kDa) differs significantly from calculated (123 kDa)

  • Apply densitometry with linear range validation

  • Normalize to total protein methods (e.g., stain-free technology) for more accurate quantification

• Immunofluorescence quantification:

  • Measure integrated intensity within defined regions of interest

  • Account for background using adjacent negative regions

  • Quantify co-localization with interacting partners using appropriate coefficients

  • When using dilutions of 1:10-1:100 , verify signal-to-noise ratio is appropriate for quantification

• Statistical considerations:

  • Determine appropriate sample sizes through power analysis

  • Use appropriate statistical tests based on data distribution

  • Account for biological vs. technical replication

• Reporting standards:

  • Document all image acquisition parameters

  • Provide raw data alongside normalized results

  • Clearly state normalization methods and reference samples

When comparing across experimental conditions, maintain consistent antibody lots, dilutions, and processing protocols to minimize technical variability.

What controls are essential when using PPP1R9A antibodies in multiplexed immunoassays?

Multiplexed immunoassays require rigorous controls:

• Antibody specificity controls:

  • Single-stain controls to verify signal specificity

  • Absorption controls using immunizing peptides if available

  • Genetic controls (knockdown/knockout) when possible

• Cross-reactivity assessment:

  • Secondary antibody-only controls

  • Isotype controls matching primary antibody host species and class

  • Test for unexpected cross-reactivity between multiplexed antibodies

• Signal separation verification:

  • Spectral unmixing controls when using fluorophores with overlapping spectra

  • Signal bleed-through assessment between channels

  • Sequential imaging controls when crosstalk is suspected

• Sample-specific controls:

  • Positive control tissues/cells with known PPP1R9A expression (PC-3, HepG2, HeLa)

  • Validation in systems where PPP1R9A is naturally enriched (neuronal synapses)

  • Internal reference standards for quantitative comparisons

For advanced multiplexed assays (CyTOF, Imaging Mass Cytometry), additional metal conjugation validation may be required to ensure PPP1R9A antibody performance is maintained after labeling.

How might PPP1R9A antibodies contribute to understanding the protein's role in oncology research?

Recent findings suggest PPP1R9A has emerging roles in cancer biology:

• Expression profiling:

  • PPP1R9A upregulation correlates with unfavorable prognosis in hepatocellular carcinoma (HCC)

  • Immunohistochemistry using validated antibodies can map expression across cancer types and stages

  • Tissue microarray analysis could identify cancer-specific patterns

• Mechanistic investigations:

  • PPP1R9A's role in cell adhesion regulation suggests involvement in metastasis

  • Co-immunoprecipitation studies can reveal cancer-specific interaction partners

  • Phospho-specific antibodies (if developed) could track activation states in tumors

• Pathway connections:

  • Gene Set Enrichment Analysis has linked PPP1R9A to MAPK, AMPK, and PI3K-Akt signaling

  • Antibody-based techniques can validate these connections at the protein level

  • Immunoprecipitation followed by mass spectrometry could identify cancer-specific complexes

• Therapeutic implications:

  • If validated as a prognostic marker, antibody-based assays could guide treatment decisions

  • Monitoring PPP1R9A levels might help assess therapy response

Researchers entering this field should employ multiple antibody-based techniques with appropriate controls to establish PPP1R9A's role in cancer biology .

What are the challenges in developing phospho-specific antibodies for PPP1R9A research?

Development of phospho-specific PPP1R9A antibodies presents significant challenges:

• Phosphorylation site identification:

  • The large difference between observed (180 kDa) and calculated (123 kDa) molecular weight suggests extensive phosphorylation

  • Mass spectrometry analysis is needed to identify functionally relevant phosphorylation sites

  • Evolutionary conservation analysis can prioritize sites for antibody development

• Technical challenges in antibody generation:

  • Phospho-epitopes may have lower immunogenicity

  • Site-specific phosphorylation may be transient

  • Multiple adjacent phosphorylation sites can complicate epitope specificity

  • Validation requires phosphatase-treated controls and phosphomimetic mutants

• Validation requirements:

  • Demonstration of specificity for phosphorylated vs. non-phosphorylated forms

  • Correlation with known regulatory events affecting PPP1R9A

  • Cross-validation with mass spectrometry or Phos-tag gels

• Application optimization:

  • Sample preparation must preserve phosphorylation status

  • Blocking and washing conditions may need optimization

  • Signal amplification may be required for low-abundance phospho-forms

Future research would benefit from collaborative approaches between protein biochemists, antibody developers, and neuroscience researchers to develop these specialized tools.