PDLIM4 Antibody, HRP conjugated

What is PDLIM4 and why is it significant in molecular biology research?

PDLIM4 (also known as LIM protein RIL or Reversion-induced LIM protein) is a scaffold protein containing one PDZ and one LIM zinc-binding domain. These domains enable it to interact with actin-associated proteins, cytoplasmic signaling molecules, and membrane receptors. PDLIM4 was initially identified as a tumor suppressor, though recent evidence suggests a more complex role that may include oncogenic properties in certain contexts. The protein plays critical roles in cytoskeletal organization, bone development, and cellular signal transduction. Understanding PDLIM4 has implications for research in cancer biology, skeletal disorders, and fundamental cellular processes .

What are the key specifications of commercially available PDLIM4 antibodies, particularly HRP-conjugated versions?

PDLIM4 antibodies are primarily available as polyclonal antibodies raised in rabbits, with the HRP-conjugated versions specifically designed for enhanced detection sensitivity. These antibodies typically show reactivity against human PDLIM4, with some cross-reactivity to mouse and rat orthologs. The antibodies target different epitopes, with immunogens ranging from amino acids 80-243 of the human PDLIM4 protein. Most HRP-conjugated versions are purified using Protein G methods and formulated in stabilized solutions containing glycerol for extended shelf life .

What is the cellular localization pattern of PDLIM4 and how does this influence experimental design?

PDLIM4 exhibits a complex localization pattern that requires careful consideration during experimental design. The protein localizes to multiple cellular compartments including cytoskeleton (particularly filamentous actin and stress fibers), early endosomes (both lumen and membrane), recycling endosomes, nucleus, and plasma membrane. Additionally, PDLIM4 can be found in specialized structures such as dendritic spines, lamellipodia, and the perinuclear region. This diverse distribution necessitates the use of co-localization studies with compartment-specific markers when investigating PDLIM4 function in specific cellular contexts .

What are the validated applications for PDLIM4 antibodies and what methodology yields optimal results?

PDLIM4 antibodies have been validated for multiple applications with specific methodological recommendations:

For HRP-conjugated PDLIM4 antibodies specifically, ELISA applications provide the most direct utility, allowing for one-step detection without secondary antibodies .

What cell and tissue types have been validated for PDLIM4 antibody applications?

PDLIM4 antibodies have been validated in diverse biological samples:

Researchers should note that expression levels vary significantly between tissue types, with epithelial and neuronal tissues often showing stronger expression. Antibody performance may therefore require optimization depending on the specific sample type being investigated .

What are the critical considerations for troubleshooting PDLIM4 antibody experiments?

When troubleshooting experiments using PDLIM4 antibodies, several critical factors should be considered:

• Antigen retrieval methods significantly impact IHC results - TE buffer at pH 9.0 is generally preferred, though citrate buffer (pH 6.0) can serve as an alternative.

• For Western blotting, sample preparation should preserve phosphorylation states, as PDLIM4 function is regulated by phosphorylation.

• Multiple bands may appear in Western blots due to potential proteolytic cleavage or post-translational modifications of PDLIM4.

• Cross-reactivity with other PDZ and LIM domain-containing proteins may occur at lower antibody dilutions.

• Storage conditions (maintaining -20°C with minimal freeze-thaw cycles) are critical for preserving antibody activity, particularly for HRP-conjugated versions .

How does PDLIM4 contribute to cytoskeletal organization and cellular motility?

PDLIM4 functions as a critical adapter protein in actin cytoskeleton remodeling and stress fiber assembly, processes essential for cellular motility and epithelial-mesenchymal transition. Through its PDZ domain, PDLIM4 interacts with actin-associated proteins to regulate filamentous actin organization, while the LIM domain facilitates interactions with signaling molecules. Recent studies in breast cancer models have demonstrated that PDLIM4 expression levels correlate with cell migratory capacity, with elevated levels associated with increased motility. This finding highlights PDLIM4's mechanistic role in cytoskeletal dynamics that directly influence cell movement and morphological changes .

What is currently understood about PDLIM4's role in skeletal disorders and osteoporosis?

PDLIM4 plays a significant role in bone development and maintenance, with genetic evidence linking it to skeletal disorders. Mutations in the PDLIM4 gene have been associated with susceptibility to osteoporosis, suggesting its importance in bone homeostasis. The molecular mechanisms involve PDLIM4's function in osteoblast development and activity, potentially through regulation of cytoskeletal organization essential for proper bone cell function and mineralization. Variation at the PDLIM4 locus represents an important determinant of osteoporosis risk, making it a potential target for therapeutic intervention in skeletal disorders .

How has our understanding of PDLIM4's role in cancer biology evolved?

• In certain breast cancer models, elevated levels of PDLIM4 correlate with increased migratory activity and higher incidence of xenograft formation and metastasis.

• The relationship between PDLIM4 and Src kinase activation appears to be context-dependent, with no consistent correlation observed across all cancer cell types.

• PDLIM4 may function differently depending on cancer type, stage, and molecular subtype.

This evolving understanding suggests that PDLIM4 may act as either a tumor suppressor or oncogene depending on the cellular context and associated signaling networks .

How should researchers address the apparent contradiction between PDLIM4's reported tumor suppressor and potential oncogenic properties?

Addressing the apparent contradiction in PDLIM4's cancer biology role requires sophisticated experimental approaches:

• Context-specific analysis: Systematically examine PDLIM4 function across multiple cancer types and molecular subtypes using both gain-of-function and loss-of-function approaches.

• Signaling pathway integration: Investigate how PDLIM4 interacts with established oncogenic and tumor suppressor pathways in specific cellular contexts, particularly focusing on its relationships with Src kinase activity.

• Post-translational modification analysis: Determine how phosphorylation and other modifications affect PDLIM4's protein interactions and functions.

• Clinical correlation studies: Analyze PDLIM4 expression patterns in patient samples with detailed clinical annotation to identify associations with disease progression and treatment responses.

• Tissue-specific knockout models: Develop conditional knockout models to assess PDLIM4's role in cancer initiation versus progression in relevant tissues .

What methodological approaches are most effective for studying PDLIM4-mediated protein interactions?

For studying PDLIM4's protein interactions, researchers should employ complementary methodological approaches:

• Domain-specific interaction mapping: Use truncated constructs to identify which PDLIM4 domains (PDZ versus LIM) mediate specific protein interactions.

• Proximity-dependent labeling: Employ BioID or APEX2 fusion proteins to identify the proximal interactome of PDLIM4 in living cells.

• Co-immunoprecipitation with phosphorylation state analysis: Combine co-IP with phospho-specific antibodies to determine how phosphorylation affects PDLIM4's interaction network.

• FRET/BRET-based interaction studies: Develop fluorescent or bioluminescent protein fusions to monitor real-time interactions in living cells.

• Cross-linking mass spectrometry: Apply protein cross-linking followed by mass spectrometry to identify direct binding partners and interaction interfaces .

What experimental design considerations are critical when investigating PDLIM4's role in cell migration and metastasis?

When investigating PDLIM4's role in cell migration and metastasis, researchers should implement these experimental design considerations:

• Physiologically relevant models: Use 3D culture systems and extracellular matrix components that better mimic in vivo conditions rather than simple 2D migration assays.

• Real-time dynamic analysis: Employ live-cell imaging with fluorescently tagged PDLIM4 to track its localization during migration events, particularly at the leading edge of migrating cells.

• Quantitative migration metrics: Apply advanced tracking algorithms to quantify multiple parameters of cell movement (velocity, directionality, persistence) rather than simple endpoint measurements.

• In vivo metastasis models: Develop appropriate animal models with modulated PDLIM4 expression to assess authentic metastatic potential, including both experimental and spontaneous metastasis approaches.

• Clinical sample validation: Correlate experimental findings with PDLIM4 expression patterns in primary tumors versus metastatic lesions from patient samples .

How can researchers effectively use PDLIM4 antibodies to study its role in specific subcellular compartments?

To effectively study PDLIM4's role in specific subcellular compartments, researchers should:

• Implement co-localization studies combining PDLIM4 antibodies with established markers for cellular compartments (early endosomes, recycling endosomes, stress fibers).

• Apply super-resolution microscopy techniques (STED, STORM, PALM) to precisely localize PDLIM4 within complex subcellular structures.

• Develop compartment-targeted PDLIM4 constructs to evaluate function in specific locations.

• Employ subcellular fractionation followed by immunoblotting to biochemically confirm PDLIM4 distribution.

• Use proximity ligation assays to verify interactions with compartment-specific proteins in situ.

• Consider using phospho-specific PDLIM4 antibodies, as phosphorylation status may influence subcellular distribution .