PHACTR4 Antibody

What is PHACTR4 and why is it important in research?

PHACTR4 (Phosphatase and Actin Regulator 4) functions as a regulator of protein phosphatase 1 (PP1) and is required for neural tube and optic fissure closure, as well as enteric neural crest cell (ENCCs) migration during development. It acts as an activator of PP1 by interacting with PPP1CA and preventing phosphorylation of PPP1CA at 'Thr-320' . More significantly, PHACTR4 has been identified as a suppressor of tumorigenesis and/or proliferation (STOP) gene that restrains normal cell proliferation . PHACTR4 suppresses proliferation and transformation in vitro and tumorigenesis in vivo, and is significantly mutated or downregulated in several cancers . This dual role in developmental processes and tumor suppression makes PHACTR4 a valuable target for antibody-based research in developmental biology, neuroscience, and cancer research.

The choice between polyclonal and monoclonal PHACTR4 antibodies depends on your specific experimental requirements:

Monoclonal antibodies recognize a single epitope, offering higher specificity and consistency between batches. The rabbit monoclonal PHACTR4 antibody [EP12543] has been validated for Western blotting, flow cytometry, and immunohistochemistry with human samples . This higher specificity makes monoclonal antibodies preferable for distinguishing between closely related proteins or specific protein variants.

For detecting endogenous levels of PHACTR4 protein, polyclonal antibodies like ABIN3186417 can be effective . If your research requires highly reproducible results across multiple experiments, the monoclonal EP12543 antibody might be more suitable .

What are the validated applications for PHACTR4 antibodies?

PHACTR4 antibodies have been validated for multiple experimental applications:

The specific application requirements may vary depending on the tissue or cell type being studied. For example, 13408-1-AP shows positive Western blot detection in mouse eye tissue, A431 cells, and human liver tissue, while positive IHC detection has been reported in human ovary cancer tissue .

How should I optimize Western blotting for PHACTR4 detection?

To optimize Western blotting for PHACTR4 detection, follow these methodological considerations:

• Sample preparation: PHACTR4 has been successfully detected in various sample types including cell lines (MCF7, Y79, A431) and tissue lysates (mouse eye, human liver) . Use appropriate lysis buffers with protease inhibitors to prevent protein degradation.

• Protein loading and molecular weight expectations: Load 20-30 μg of total protein per lane. While the calculated molecular weight of PHACTR4 is 78 kDa, the observed molecular weight ranges from 70-78 kDa . This discrepancy could be due to post-translational modifications or protein isoforms.

• Antibody dilutions:

  • Primary antibody: Use 1:500-1:1000 dilution for most PHACTR4 antibodies

  • Secondary antibody: For HRP-conjugated anti-rabbit IgG, a 1:1000-1:5000 dilution is typically recommended

• Blocking and incubation conditions: Use 5% non-fat milk or BSA in TBST for blocking. Incubate with primary antibody overnight at 4°C for optimal results.

• Detection controls: MCF7 and Y79 cell lysates have been used as positive controls with antibody ab192882, showing a band at approximately 70 kDa .

What are the recommended protocols for immunohistochemistry with PHACTR4 antibodies?

For immunohistochemistry with PHACTR4 antibodies, consider the following protocol recommendations:

• Tissue preparation: Use appropriately fixed (typically 10% neutral buffered formalin) and paraffin-embedded tissue sections. Human brain tissue has been successfully used with ab192882 .

• Antigen retrieval methods:

  • For ab192882: Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0

  • For 13408-1-AP: Suggested antigen retrieval with TE buffer pH 9.0, or alternatively with citrate buffer pH 6.0

• Antibody dilutions:

  • 13408-1-AP: Use at 1:50-1:500 dilution

  • ab192882: Use at 1:2000 dilution

• Detection systems: For ab192882, prediluted HRP Polymer for Rabbit/Mouse IgG has been successfully used as a secondary detection system .

• Counterstaining: Hematoxylin can be used for nuclear counterstaining .

• Positive controls: Human brain tissue has shown positive staining with ab192882, while human ovary cancer tissue has been positive with 13408-1-AP .

• Negative controls: Always include appropriate negative controls such as sections incubated without primary antibody or with isotype control antibodies .

What is the subcellular localization of PHACTR4 and how can it be visualized?

PHACTR4 exhibits specific subcellular localization patterns that can be visualized using immunofluorescence techniques. During neural development, PHACTR4 localizes to the ventral neural tube where it activates PP1, leading to down-regulation of cell proliferation within cranial neural tissue and the neural retina .

For visualizing PHACTR4's subcellular localization:

• Cell fixation: Use 2% paraformaldehyde for cell fixation, as demonstrated in flow cytometry protocols with SH-SY5Y cells .

• Immunofluorescence protocol:

  • For 13408-1-AP: Use at 1:20-1:200 dilution for IF/ICC applications

  • A431 cells have shown positive staining with PHACTR4 antibodies in IF/ICC applications

• Co-localization studies: Consider co-staining with markers for protein phosphatase 1 (PP1) or actin to examine PHACTR4's functional interactions, as PHACTR4 is known to interact with PP1 and regulate actin dynamics .

• Confocal microscopy: High-resolution confocal microscopy is recommended for detailed subcellular localization studies.

Understanding PHACTR4's subcellular distribution can provide insights into its functional role in different cellular contexts, particularly in neural development and tumor suppression.

How can I study PHACTR4's role as a tumor suppressor in my research?

To investigate PHACTR4's role as a tumor suppressor gene (STOP gene), consider the following methodological approaches:

• Expression analysis in normal vs. cancer tissues:

  • Use PHACTR4 antibodies in Western blotting or IHC to compare expression levels between normal and cancer tissues

  • PHACTR4 has been found to be significantly mutated or downregulated in several cancers

• Functional studies:

  • Knockdown experiments: Use siRNA or shRNA to reduce PHACTR4 expression and assess effects on cell proliferation, transformation, and tumorigenic potential

  • Overexpression experiments: Reintroduce PHACTR4 in cancer cell lines with low endogenous expression to assess if this limits proliferation and tumor growth

• Mechanistic investigations:

  • Examine PHACTR4's interaction with PP1 using co-immunoprecipitation with PHACTR4 antibodies

  • Investigate the phosphorylation status of PPP1CA at 'Thr-320' in the presence and absence of PHACTR4

  • Assess the activation status of downstream targets like cofilin (COF1 or COF2)

• In vivo tumor models:

  • Generate xenograft models using cells with manipulated PHACTR4 expression

  • Research has shown that reintroduction of Phactr4 limits tumor growth in cancer cells

• Clinical correlation studies:

  • Correlate PHACTR4 expression levels (detected by antibodies) with patient outcomes and clinicopathological features

These approaches can help elucidate PHACTR4's role in suppressing tumorigenesis and identify potential therapeutic strategies targeting this pathway.

How do I address non-specific binding or background issues with PHACTR4 antibodies?

When encountering non-specific binding or high background with PHACTR4 antibodies, consider the following troubleshooting strategies:

• Antibody dilution optimization:

  • Try a series of dilutions to determine the optimal concentration

  • For Western blotting, 1:500-1:1000 is recommended for most PHACTR4 antibodies

  • For IHC, more dilute solutions (1:50-1:500 for 13408-1-AP or 1:2000 for ab192882) may reduce background

• Blocking optimization:

  • Extend blocking time (1-2 hours at room temperature)

  • Try different blocking agents (5% BSA vs. 5% non-fat milk)

  • Add 0.1-0.3% Triton X-100 to blocking buffer for membrane permeabilization in IF applications

• Washing stringency:

  • Increase the number and duration of washing steps

  • Use TBST with 0.1-0.3% Tween-20 for more stringent washing

• Sample preparation improvements:

  • Ensure complete protein denaturation for Western blotting

  • For IHC/ICC, optimize fixation conditions and antigen retrieval methods

  • For 13408-1-AP in IHC, try both recommended antigen retrieval methods: TE buffer pH 9.0 or citrate buffer pH 6.0

• Controls to include:

  • Negative controls: Samples known not to express PHACTR4

  • Isotype controls: Rabbit monoclonal IgG for ab192882

  • Antibody omission controls: Sections/cells processed without primary antibody

• Evaluating specificity:

  • Confirm the observed molecular weight (70-78 kDa for PHACTR4)

  • Consider peptide competition assays to confirm antibody specificity

How can I interpret variations in PHACTR4 molecular weight across different samples?

PHACTR4 has a calculated molecular weight of 78 kDa (702 amino acids), but the observed molecular weight ranges from 70-78 kDa . These variations may be biologically significant and should be carefully interpreted:

• Possible explanations for molecular weight variations:

  • Post-translational modifications: Phosphorylation, glycosylation, or other modifications can alter molecular weight

  • Alternative splicing: Different isoforms may exist in different tissues or cellular contexts

  • Proteolytic processing: PHACTR4 might undergo specific cleavage in certain conditions

  • Species differences: Slight variations may occur between human and mouse PHACTR4

• Analytical approaches to characterize variations:

  • Phosphatase treatment: To determine if variations are due to phosphorylation

  • 2D gel electrophoresis: To separate proteins based on both molecular weight and isoelectric point

  • Mass spectrometry: For precise identification of modifications or variants

  • Isoform-specific antibodies: If available, these can help distinguish between different forms

• Biological significance considerations:

  • Different molecular weight forms may have distinct functions or subcellular localizations

  • Certain cancer types may show specific alterations in PHACTR4 processing or modification

  • The tumor suppressor function may be associated with specific forms of PHACTR4

• Reporting standards:

  • Always report both the expected (calculated) and observed molecular weights

  • Note any sample-specific variations in molecular weight

  • Include loading controls to ensure equal protein loading and transfer

How can I investigate PHACTR4's interaction with protein phosphatase 1 (PP1)?

PHACTR4 functions as a regulator of protein phosphatase 1 (PP1), and investigating this interaction is crucial for understanding its biological roles:

• Co-immunoprecipitation (Co-IP) strategies:

  • Use PHACTR4 antibodies to pull down PHACTR4 complexes and probe for PP1 (PPP1CA)

  • Alternatively, use PP1 antibodies for immunoprecipitation and detect PHACTR4 in the precipitate

  • Include appropriate controls: IgG control, input samples, and lysates from cells with PHACTR4 knockdown

• Functional assays:

  • Phosphatase activity assays to measure PP1 activity in the presence and absence of PHACTR4

  • Monitor the phosphorylation status of PPP1CA at 'Thr-320', as PHACTR4 prevents phosphorylation at this site

  • Examine downstream targets like cofilin (COF1 or COF2) that are dephosphorylated and activated through PHACTR4-mediated PP1 activation

• Structural studies:

  • Map the interaction domains using truncated protein constructs

  • Consider computational modeling of the PHACTR4-PP1 interaction

• Subcellular localization:

  • Use double immunofluorescence with PHACTR4 and PP1 antibodies to assess co-localization

  • Examine changes in localization under different cellular conditions

• Effect of PHACTR4 mutations:

  • Analyze how cancer-associated mutations in PHACTR4 affect its interaction with PP1

  • This can provide insights into how PHACTR4 mutations might contribute to tumorigenesis

What are the cutting-edge approaches for studying PHACTR4 in neural development and cancer?

Current advanced research on PHACTR4 focuses on its dual roles in neural development and tumor suppression:

• In neural development research:

  • Conditional knockout models: Tissue-specific and temporally controlled deletion of PHACTR4 in neural tissues

  • Live imaging techniques: To visualize the migration of enteric neural crest cells (ENCCs) in the presence and absence of PHACTR4

  • Single-cell transcriptomics: To map PHACTR4 expression patterns during neural development

  • CRISPR/Cas9 genome editing: To introduce specific mutations that affect PHACTR4's interaction with PP1 and study the consequences for neural tube closure

• In cancer research:

  • Patient-derived xenografts (PDXs): To study the effect of PHACTR4 restoration in tumors with low PHACTR4 expression

  • Analysis of PHACTR4 mutations in cancer genomics databases: To identify recurrent mutations and correlate with clinical outcomes

  • Therapeutic approaches: Development of peptides or small molecules that could restore PHACTR4 function in cancers where it is downregulated

  • Combination therapy studies: Investigating whether restoring PHACTR4 function can enhance sensitivity to conventional therapies

• Multidisciplinary approaches:

  • Integration of phosphoproteomics: To identify the complete set of proteins affected by PHACTR4-mediated PP1 activation

  • Systems biology: Modeling the PHACTR4-PP1-actin regulatory network and its perturbation in cancer

  • Translational research: Evaluating PHACTR4 as a biomarker for cancer prognosis or treatment response

• Technical innovations:

  • Proximity labeling techniques (BioID, APEX): To identify novel PHACTR4 interacting partners in different cellular contexts

  • Super-resolution microscopy: For detailed visualization of PHACTR4's subcellular localization and co-localization with interaction partners

  • Organoid models: To study PHACTR4 function in 3D tissue-like structures that better recapitulate in vivo conditions

These cutting-edge approaches can provide deeper insights into PHACTR4's functions and potentially lead to new therapeutic strategies targeting PHACTR4-dependent pathways in developmental disorders and cancer.

What are the emerging research areas for PHACTR4 antibodies?

The field of PHACTR4 research is evolving, with several promising directions for antibody applications:

• Biomarker development: PHACTR4 antibodies could be valuable for assessing PHACTR4 expression in patient samples as a potential prognostic or predictive biomarker in cancers where PHACTR4 is frequently downregulated .

• Therapeutic monitoring: For experimental therapies aimed at restoring PHACTR4 expression or function, antibodies would be essential tools to monitor treatment efficacy.

• Isoform-specific detection: Development of antibodies that can distinguish between different PHACTR4 isoforms or post-translationally modified forms would advance our understanding of PHACTR4's diverse functions.

• Multiplex imaging: Integration of PHACTR4 antibodies into multiplex immunofluorescence panels to study its expression in relation to other proteins in the PP1 regulatory pathway within the spatial context of tissues.

• Single-cell applications: Adaptation of PHACTR4 antibodies for single-cell protein analysis techniques to understand cell-to-cell variability in PHACTR4 expression and function.