PLEKHA5 Antibody

What is PLEKHA5 and what are its key molecular characteristics?

PLEKHA5 (pleckstrin homology domain containing, family A member 5) is a protein with a calculated molecular weight of approximately 127 kDa (1116 amino acids) . Interestingly, the observed molecular weight in laboratory analyses has shown two distinct bands at 127 kDa and 74 kDa, suggesting possible post-translational modifications or alternative splicing variants . The protein contains pleckstrin homology domains that typically mediate protein-protein interactions and may be involved in intracellular signaling. PLEKHA5's gene ID is 54477 according to NCBI databases, and its UniProt ID is Q9HAU0 .

What types of PLEKHA5 antibodies are currently available for research?

Multiple PLEKHA5 antibodies are available targeting different epitopes of the protein:

Most available antibodies are rabbit polyclonal antibodies, which offer high sensitivity but may have batch-to-batch variation . The antibodies target various regions including N-terminal, C-terminal, and internal domains, allowing researchers to select the most appropriate antibody based on their experimental needs and the specific domain of interest.

What are the validated applications for PLEKHA5 antibodies?

PLEKHA5 antibodies have been validated for several research applications:

• Western Blotting (WB): Most PLEKHA5 antibodies are validated for WB at dilutions ranging from 1:500-1:1000 .

• Enzyme-Linked Immunosorbent Assay (ELISA): Several antibodies demonstrate reactivity in ELISA applications .

• Immunohistochemistry (IHC): Some antibodies like HPA035923 are validated for IHC at dilutions of 1:500-1:1000 .

• Immunoprecipitation (IP): Certain antibodies are specifically validated for IP applications .

When designing experiments, researchers should note that optimal dilutions may need to be determined empirically for each specific experimental system and sample type to obtain optimal results .

What are the recommended protocols for Western blotting using PLEKHA5 antibodies?

For Western blotting with PLEKHA5 antibodies, the following methodological considerations are critical:

• Sample Preparation: PLEKHA5 has been successfully detected in various tissue types including mouse testis, human heart, and mouse kidney tissues . Proper lysis buffers containing protease inhibitors are essential.

• Dilution Optimization: Start with the recommended dilution range (1:500-1:1000) , but be prepared to optimize based on your specific sample type and protein expression levels.

• Detection System: Most PLEKHA5 antibodies are unconjugated and require appropriate secondary antibodies for detection.

• Expected Bands: Look for bands at approximately 127 kDa and 74 kDa, as these are the observed molecular weights for PLEKHA5 . The presence of multiple bands may indicate alternative splicing or post-translational modifications.

• Controls: Include positive controls such as mouse testis, human heart, or mouse kidney tissue lysates, which have been validated to express PLEKHA5 .

Each antibody may have specific optimized protocols, and manufacturers often provide detailed Western blotting protocols tailored to their specific antibody products .

How should PLEKHA5 antibodies be stored to maintain optimal reactivity?

For optimal performance and longevity of PLEKHA5 antibodies, proper storage conditions are crucial:

Always refer to the specific storage instructions provided by the manufacturer for each antibody product, as formulations may vary.

What is the significance of PLEKHA5 in melanoma brain metastasis research?

PLEKHA5 has emerged as a potentially important biomarker and mediator in melanoma brain metastasis:

• Association with Brain Metastases: PLEKHA5 expression in melanoma tumors has been significantly associated with early development of brain metastases . Approximately 40% of patients with metastatic melanoma develop brain metastases, making this a clinically relevant area of investigation.

• Differential Expression: Gene expression profiling studies have identified PLEKHA5 as differentially expressed in both cell line models of brain metastasis (cerebrotropic A375Br cells versus parental A375P cells) and in patient samples .

• Prognostic Value: At the protein level, quantitative immunofluorescence studies using tissue microarrays containing 169 metastatic melanoma cases demonstrated that PLEKHA5 expression is associated with decreased brain metastasis-free survival .

• Site Specificity: Interestingly, PLEKHA5 overexpression was not associated with metastasis to other sites, suggesting a specific role in brain tropism .

This research suggests that PLEKHA5 may serve both as a biomarker to identify patients at risk for brain metastases and potentially as a therapeutic target to prevent or treat brain metastases in melanoma patients.

What experimental approaches have been used to study PLEKHA5's role in brain metastasis?

Several sophisticated experimental approaches have been employed to investigate PLEKHA5's function in brain metastasis:

• Gene Expression Profiling: Comparative gene expression analysis between brain metastatic cells (A375Br) and parental cells (A375P), as well as between patient samples with early brain metastases versus those without .

• Tissue Microarray Analysis: A tissue microarray containing 169 metastatic melanoma cases with variable time to brain metastasis was analyzed using quantitative immunofluorescence to correlate PLEKHA5 protein expression with clinical outcomes .

• In Vitro Blood-Brain Barrier (BBB) Model: Researchers developed an in vitro model of the BBB to evaluate PLEKHA5's potential role in facilitating melanoma cell passage across this critical barrier .

• Knockdown Studies: PLEKHA5 was knocked down in A375Br cells (brain-metastatic) and YUMUL cells (derived from a patient with overwhelming brain metastases) to assess effects on:

  • Cell viability

  • BBB transmigration capabilities

  • Invasion potential

• Comparative Analysis: The effects of PLEKHA5 knockdown were compared between brain-metastatic cells (A375Br) and parental cells (A375P), revealing selective effects on the viability of brain-metastatic cells .

These methodological approaches provide a framework for researchers investigating the roles of other proteins in cancer metastasis, particularly in the context of brain tropism.

How does PLEKHA5 knockdown affect melanoma cell behavior in experimental models?

PLEKHA5 knockdown experiments have revealed several important functional consequences:

• Selective Viability Effects: Knockdown of PLEKHA5 significantly decreased the viability of brain-metastatic A375Br cells, but interestingly did not affect the viability of parental A375P cells . This suggests a selective dependency of brain-metastatic cells on PLEKHA5 function.

• BBB Transmigration Inhibition: PLEKHA5 knockdown inhibited the ability of melanoma cells to transmigrate across an in vitro BBB model . This finding suggests that PLEKHA5 may play a specific role in facilitating the passage of melanoma cells across the BBB, a critical step in brain metastasis formation.

• Invasion Suppression: Reduced PLEKHA5 expression also inhibited invasion capabilities in vitro , suggesting a role in the invasive phenotype of melanoma cells.

• Consistent Effects Across Models: Similar results were observed with YUMUL cells, which were cultured from a patient with overwhelming brain metastases , providing further validation of these findings across different cellular models.

These experimental findings suggest that PLEKHA5 may represent a potential therapeutic target to specifically prevent or reduce brain metastases in melanoma patients, as its inhibition might decrease passage across the BBB and reduce the survival and proliferation of melanoma cells in the brain microenvironment.

What controls should be included when validating PLEKHA5 antibody specificity?

Proper validation of PLEKHA5 antibody specificity requires several controls:

• Positive Tissue Controls: Include known positive tissues such as mouse testis, human heart, and mouse kidney tissues, which have been validated to express PLEKHA5 .

• Knockdown/Knockout Controls: When possible, include samples where PLEKHA5 has been knocked down or knocked out using siRNA, shRNA, or CRISPR-Cas9 technology. This is the gold standard for antibody validation.

• Peptide Competition: Perform peptide competition assays using the immunizing peptide to confirm specificity. For instance, with antibodies generated against synthetic peptides (like ABIN2791857 targeting the C-terminal sequence "ASDQSPLQSP SNLRDNPFRT TQTRRRDDKE LDTAIRENDV KPDHETPATE"), pre-incubation with excess peptide should abolish specific signals .

• Multiple Antibodies: Validate findings using multiple antibodies targeting different epitopes of PLEKHA5 to ensure consistency in detection and localization patterns.

• Cross-Reactivity Assessment: Test antibodies on samples from multiple species to confirm the predicted cross-reactivity. For example, ABIN2791857 has predicted reactivity with Dog (86%), Human (100%), and Mouse (92%) .

These comprehensive validation steps ensure that experimental observations genuinely reflect PLEKHA5 biology rather than non-specific antibody interactions.

How can researchers troubleshoot weak or absent PLEKHA5 signal in Western blotting?

When encountering weak or absent PLEKHA5 signal in Western blotting, consider the following methodological troubleshooting steps:

• Sample Preparation Optimization:

  • Ensure complete protein extraction using appropriate lysis buffers

  • Include fresh protease inhibitors to prevent degradation

  • Verify protein concentration using reliable quantification methods

• Antibody Concentration Adjustment:

  • Try a more concentrated antibody dilution than the recommended 1:500-1:1000 range

  • Extend primary antibody incubation time (overnight at 4°C)

• Protein Loading:

  • Increase the amount of total protein loaded (50-100 μg may be necessary for low-abundance proteins)

  • Verify transfer efficiency using reversible staining methods (Ponceau S)

• Detection System Enhancement:

  • Use more sensitive detection substrates (enhanced chemiluminescence plus)

  • Consider amplification systems or more sensitive secondary antibodies

  • Extend exposure time when imaging

• Buffer and Blocking Optimization:

  • Test different blocking reagents (BSA vs. milk)

  • Optimize salt concentration in wash buffers

  • Consider adding detergents like 0.1% Tween-20 to reduce background

• Alternative Antibody Selection:

  • If one antibody consistently fails, try another antibody targeting a different epitope

  • Consider the observed molecular weights (127 kDa and 74 kDa) when analyzing results

These systematic troubleshooting approaches can help overcome technical challenges in detecting PLEKHA5 protein in Western blotting applications.

How should researchers interpret varying PLEKHA5 expression patterns across different cancer models?

Interpreting PLEKHA5 expression patterns across cancer models requires careful consideration of several factors:

• Tissue-Specific Expression: PLEKHA5 may have tissue-specific expression patterns. When comparing expression across models, consider the tissue of origin and baseline expression levels in normal counterparts.

• Multiple Molecular Forms: The observation of PLEKHA5 at both 127 kDa and 74 kDa suggests the existence of multiple isoforms or post-translational modifications. Different cancer models may express different forms, potentially with distinct functions.

• Metastatic Site Specificity: PLEKHA5 overexpression has been specifically associated with brain metastasis but not other metastatic sites in melanoma . When examining other cancer types, researchers should specifically analyze metastatic patterns in relation to PLEKHA5 expression.

• Functional Context: Interpret PLEKHA5 expression in light of functional data. For example, PLEKHA5 knockdown affected the viability of brain-metastatic A375Br cells but not parental A375P cells , suggesting context-dependent functions.

• Clinical Correlation: Validate findings from cell models with patient data whenever possible. The association between PLEKHA5 expression and decreased brain metastasis-free survival in melanoma patients provides strong clinical relevance to observations in model systems.

These interpretative frameworks help researchers contextualize PLEKHA5 expression patterns within the broader landscape of cancer biology and metastasis.

What considerations should be made when designing PLEKHA5 knockdown or overexpression experiments?

When designing PLEKHA5 knockdown or overexpression experiments, researchers should consider:

• Target Specificity:

  • Design multiple siRNAs or shRNAs targeting different regions of PLEKHA5 mRNA

  • Include scrambled controls with similar GC content

  • Validate knockdown efficiency at both mRNA and protein levels

• Expression Vector Selection for Overexpression:

  • Consider using vectors with different promoters for varying expression levels

  • Include tags (FLAG, HA, etc.) that don't interfere with PLEKHA5 function

  • Verify that the construct includes the full 1116 amino acid sequence for full-length protein expression

• Cell Line Selection:

  • Include both brain-metastatic lines (like A375Br, YUMUL) and non-brain-metastatic lines (like A375P)

  • Consider using patient-derived cell lines when available

  • Include normal cell lines as controls for specificity

• Functional Readouts:

  • Measure cell viability and proliferation

  • Assess BBB transmigration abilities using in vitro models

  • Evaluate invasion potential

  • Consider in vivo models for metastasis studies

• Temporal Considerations:

  • Analyze both immediate and long-term effects of PLEKHA5 modulation

  • Consider inducible systems for temporal control of expression

• Downstream Analysis:

  • Examine effects on known signaling pathways

  • Perform RNA-seq or proteomic analysis to identify affected pathways

  • Validate key findings using orthogonal approaches

These design considerations ensure robust and reproducible experiments that provide meaningful insights into PLEKHA5 function in cancer biology and metastasis.

What emerging technologies might enhance PLEKHA5 research in cancer and metastasis?

Several cutting-edge technologies hold promise for advancing PLEKHA5 research:

• CRISPR-Cas9 Genome Editing:

  • Generation of PLEKHA5 knockout cell lines and animal models

  • Domain-specific mutations to identify critical functional regions

  • CRISPRi/CRISPRa systems for reversible modulation of expression

• Single-Cell Analysis:

  • Single-cell RNA-seq to identify PLEKHA5-expressing subpopulations within heterogeneous tumors

  • Single-cell proteomics to correlate PLEKHA5 protein levels with other signaling molecules

  • Spatial transcriptomics to map PLEKHA5 expression within the tumor microenvironment

• Advanced BBB Models:

  • Microfluidic organ-on-chip BBB models with patient-derived cells

  • 3D spheroid models incorporating endothelial cells, pericytes, and astrocytes

  • Real-time imaging of cancer cell-BBB interactions

• Proximity Labeling Techniques:

  • BioID or APEX2 fusion proteins to identify PLEKHA5 interaction partners

  • Analysis of PLEKHA5 protein complexes in brain-metastatic versus non-brain-metastatic contexts

• In Vivo Metastasis Imaging:

  • Intravital microscopy to visualize PLEKHA5-expressing cells during metastasis

  • PET imaging with PLEKHA5-targeted tracers for clinical translation

These technological approaches can provide deeper mechanistic insights into PLEKHA5's role in cancer metastasis and potentially identify novel therapeutic strategies.

How might PLEKHA5 research translate into clinical applications for melanoma patients?

PLEKHA5 research holds several potential translational applications for melanoma patients:

• Prognostic Biomarker Development:

  • Development of standardized IHC protocols for PLEKHA5 detection in clinical samples

  • Prospective studies correlating PLEKHA5 expression with brain metastasis risk

  • Integration with other biomarkers for improved risk stratification

• Therapeutic Target Exploration:

  • Small molecule inhibitors targeting PLEKHA5 protein or its interactions

  • Peptide-based inhibitors of PLEKHA5-mediated BBB transmigration

  • Antibody-drug conjugates targeting PLEKHA5-expressing cells

• Prevention Strategies:

  • Prophylactic treatments for high-PLEKHA5 expressing melanomas to prevent brain metastasis

  • Combination with existing therapies for enhanced BBB penetration

  • Development of brain-targeted drug delivery systems

• Monitoring Treatment Response:

  • Liquid biopsy approaches to monitor PLEKHA5 expression in circulating tumor cells

  • Correlation of PLEKHA5 levels with response to systemic or targeted therapies

  • Early detection of brain metastasis development

• Clinical Trial Design:

  • Stratification of patients based on PLEKHA5 expression in clinical trials

  • Targeted enrollment of high-PLEKHA5 expressors for brain metastasis prevention studies

  • Development of companion diagnostics for PLEKHA5-targeted therapies

The association between PLEKHA5 expression and brain metastasis provides a strong rationale for these translational approaches, potentially benefiting the approximately 40% of metastatic melanoma patients who develop brain metastases.