PLEKHA7 Antibody, HRP conjugated

What is PLEKHA7 and why is it significant in epithelial tissue research?

PLEKHA7 (Pleckstrin Homology Domain Containing Family A Member 7) is a protein that links the E-cadherin-p120 ctn complex to the microtubule cytoskeleton. Its significance stems from its specific localization at the adherens junction belt in epithelial tissues. PLEKHA7 contains two WW domains and one pleckstrin homology (PH) domain in its N-terminal half, and coiled-coil (cc) and proline-rich domains in its C-terminal half . This unique protein has been detected in multiple epithelial tissues including kidney, liver, pancreas, intestine, retina, and cornea, but notably demonstrates a distinct subcellular localization compared to other junction proteins like ZO-1 . Unlike many adherens junction markers that distribute along the lateral regions of polarized epithelial cells, PLEKHA7 primarily concentrates at the apical junctional belt, similar to afadin . This specific localization pattern makes PLEKHA7 antibodies particularly valuable for investigating epithelial junction architecture and integrity in various tissues.

What are the known molecular isoforms of PLEKHA7 that researchers should be aware of when selecting antibodies?

Researchers should be aware that PLEKHA7 exists in multiple isoforms across different tissues, which may affect antibody binding and experimental outcomes. Northern blot analysis has identified two major PLEKHA7 transcripts with apparent sizes of approximately 5.5 kb and 6.5 kb in brain, kidney, liver, small intestine, placenta, and lung, while heart tissue predominantly expresses the 5.5 kb transcript . At the protein level, immunoblotting of epithelial tissue lysates reveals major polypeptides of Mr ~135-145 kDa . Bioinformatic analysis predicts two isoforms for human PLEKHA7 (both approximately 127 kDa) and five isoforms for mouse PLEKHA7 (approximately 127, 144, 115, 117, and 107 kDa) . When designing experiments, researchers should consider these tissue-specific expression patterns and molecular weight variations to properly interpret results obtained with PLEKHA7 antibodies.

What are the technical advantages of using HRP-conjugated PLEKHA7 antibodies over unconjugated versions?

HRP-conjugated PLEKHA7 antibodies offer several methodological advantages for researchers. The direct conjugation eliminates the need for secondary antibodies in detection workflows, which reduces background signal, minimizes cross-reactivity issues, and shortens experimental protocols. This is particularly valuable when working with complex tissue samples or when performing multiplexed experiments. HRP conjugation provides enhanced sensitivity through enzymatic signal amplification, allowing detection of low-abundance PLEKHA7 proteins in samples where expression might be reduced, such as in lens epithelial cells and iris tissue of PACG patients where PLEKHA7 has been shown to be downregulated . Additionally, HRP-conjugated antibodies have greater stability and longer shelf-life compared to fluorophore-conjugated antibodies, making them more suitable for laboratories with intermittent PLEKHA7 research projects.

How should researchers optimize Western blotting protocols when using HRP-conjugated PLEKHA7 antibodies?

For optimal Western blotting results with HRP-conjugated PLEKHA7 antibodies, researchers should implement the following methodological considerations:

• Sample preparation: Extract proteins from epithelial tissues using buffers containing protease inhibitors to prevent degradation of the high molecular weight PLEKHA7 isoforms (135-145 kDa).

• Gel selection: Use 8% SDS-PAGE gels to achieve proper separation of the large PLEKHA7 isoforms. Published research demonstrates effective separation with this percentage .

• Transfer optimization: Perform wet transfer at low voltage (30V) overnight at 4°C to ensure complete transfer of high molecular weight PLEKHA7 proteins.

• Blocking conditions: Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature to reduce background without interfering with antibody binding.

• Antibody dilution: Based on published protocols, dilute HRP-conjugated PLEKHA7 antibodies 1:100-1:10,000 depending on the specific antibody and application, as demonstrated in studies using polyclonal anti-PLEKHA7 antibodies at 1:10,000 for immunoblotting .

• Controls: Include positive controls (e.g., lysates from kidney epithelial cells like MDCK or mpkCCDc14) and negative controls (PLEKHA7-depleted cells via shRNA knockdown) .

• Detection optimization: Use enhanced chemiluminescence with shorter exposure times to prevent signal saturation, as HRP enzymatic activity can rapidly generate strong signals.

What are the recommended protocols for immunofluorescence studies targeting PLEKHA7 in different epithelial tissues?

For immunofluorescence studies targeting PLEKHA7 in epithelial tissues, researchers should follow these tissue-specific recommendations:

General protocol for all tissues:

• Fix samples in 4% paraformaldehyde for 15-20 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 for 10 minutes

• Block with 3% BSA in PBS for 1 hour at room temperature

• If using the HRP-conjugated antibody for immunofluorescence, convert to fluorescence using tyramide signal amplification

• For co-localization studies, consider combinations with markers such as p120 ctn, β-catenin, E-cadherin, or afadin

Tissue-specific considerations:

When examining co-localization patterns, note that PLEKHA7 shows distinct distribution compared to other junction proteins - it is concentrated at apical junctional belts but not along lateral regions of polarized epithelial cells .

What approaches should be used for validating PLEKHA7 antibody specificity in experimental systems?

To validate PLEKHA7 antibody specificity, researchers should implement a comprehensive validation strategy including:

• Western blot validation: Compare the molecular weight of detected bands with expected PLEKHA7 isoforms (~135-145 kDa) across different tissue types . Confirm that the antibody detects the recombinant antigen used for immunization.

• RNA interference controls: Use shRNA-mediated knockdown of PLEKHA7 to confirm signal reduction in both Western blot and immunofluorescence applications. This approach has been successfully implemented in MDCK cells to validate antibody specificity .

• Epitope mapping: If working with monoclonal antibodies, determine the specific epitope recognized by the antibody. Previous studies have mapped epitopes of anti-PLEKHA7 monoclonal antibodies to within residues 920-1020 using bacterially expressed fragments .

• Cross-reactivity assessment: Test the antibody against related proteins, particularly other PLEKHA family members, to ensure specificity. Protein G purified antibodies with >95% purity should show minimal cross-reactivity .

• Immunoprecipitation validation: Verify that the antibody can immunoprecipitate the native protein from cell lysates, as this confirms recognition of the properly folded protein.

• Comparison across species: If the antibody is expected to be cross-reactive, validate its specificity across relevant species (e.g., human, mouse, dog) using appropriate positive control samples like mpkCCDc14 (mouse) and MDCK (dog) cells .

How can PLEKHA7 antibodies be used to investigate its role in primary angle closure glaucoma (PACG)?

PLEKHA7 antibodies provide crucial tools for investigating the molecule's role in PACG pathogenesis through several advanced research approaches:

• Expression correlation studies: PLEKHA7 antibodies can be used to quantify protein expression in lens epithelial cells and iris tissue samples from PACG patients compared to controls. Research has shown that PLEKHA7 is downregulated in these tissues from PACG patients .

• Genotype-phenotype correlation: Researchers can use PLEKHA7 antibodies to measure protein expression levels in patients with different genotypes at the rs11024102 SNP locus. Studies have demonstrated that carriers of the C risk allele show significantly reduced PLEKHA7 expression compared to non-risk allele carriers .

• Blood-aqueous barrier (BAB) integrity assessment: Since PLEKHA7 has been identified as a regulator of BAB function, researchers can use antibodies to examine its expression and localization in components of the BAB in PACG models, correlating changes with barrier integrity measurements.

• Mechanistic studies: PLEKHA7 antibodies can be employed to investigate the protein's novel Rac1/Cdc42 GAP activity in ocular tissues, which affects actin cytoskeleton organization and paracellular barrier integrity . This can be done through co-immunoprecipitation experiments to assess PLEKHA7 interactions with GTP-bound Rac1 and Cdc42.

• Therapeutic target validation: By monitoring PLEKHA7 expression and localization during experimental manipulation of its expression or activity, researchers can evaluate its potential as a therapeutic target for PACG.

What is the significance of PLEKHA7's subcellular localization in understanding its function, and how can antibodies help elucidate this?

The precise subcellular localization of PLEKHA7 is crucial for understanding its function, and antibodies provide essential tools for elucidating these patterns:

Understanding these localization patterns through antibody-based studies provides critical insights into PLEKHA7's functional integration with junction complexes and cytoskeletal elements.

How can researchers utilize PLEKHA7 antibodies to investigate its novel Rac1/Cdc42 GAP activity in different cellular contexts?

Researchers can employ PLEKHA7 antibodies to investigate its GAP activity through several methodological approaches:

• Co-immunoprecipitation studies: Use PLEKHA7 antibodies for pull-down experiments to identify specific interactions with GTP-bound Rac1 and Cdc42, but not RhoA, as demonstrated in previous research . This approach confirms binding specificity to active GTPases.

• GTPase activity assays: Following PLEKHA7 knockdown or overexpression, use antibodies to confirm altered PLEKHA7 levels while measuring corresponding changes in Rac1/Cdc42 GTP hydrolysis rates. Previous studies have shown that PLEKHA7 stimulates GTP hydrolysis without affecting nucleotide exchange .

• Cell migration analysis with immunofluorescence: Track changes in actin cytoskeleton organization (which is affected by Rac1/Cdc42 activity) following PLEKHA7 manipulation, using antibodies to confirm expression levels. This approach has been successful in human immortalized non-pigmented ciliary epithelium (h-iNPCE) and primary trabecular meshwork cells .

• Barrier function correlation: Measure paracellular barrier integrity in epithelial monolayers following PLEKHA7 silencing, using antibodies to confirm knockdown efficiency. Research has shown that PLEKHA7 silencing compromises the paracellular barrier between h-iNPCE cells through its Rac1/Cdc42 GAP activity .

• Tissue-specific GAP activity investigation: Compare PLEKHA7's GAP activity across different tissues relevant to PACG pathogenesis (iris, lens epithelium, ciliary epithelium) using tissue-specific expression systems and antibody-based confirmation.

The table below summarizes experimental approaches for investigating PLEKHA7 GAP activity:

What are common technical challenges when using HRP-conjugated PLEKHA7 antibodies, and how can researchers address them?

Researchers frequently encounter several technical challenges when using HRP-conjugated PLEKHA7 antibodies. Here are evidence-based solutions:

• High background signal in Western blots:

  • Increase blocking time to 2 hours using 5% BSA instead of milk

  • Dilute the HRP-conjugated antibody further (1:15,000-1:20,000)

  • Include 0.05% Tween-20 in all washing steps

  • Use freshly prepared buffers to prevent bacterial growth that can react with HRP

• Multiple bands or unexpected molecular weights:

  • Consider PLEKHA7 isoform expression (expected bands at ~135-145 kDa)

  • Be aware of potential cross-reacting proteins, such as the ~240 kDa polypeptide observed in pancreatic tissue that could not be immunoprecipitated

  • Include positive controls from tissues with known PLEKHA7 expression (kidney epithelial cells)

  • Use gradient gels (4-12%) to better resolve high molecular weight proteins

• Poor signal in immunohistochemistry:

  • Optimize antigen retrieval (citrate buffer pH 6.0, 20 minutes at 95°C)

  • Extend primary antibody incubation to overnight at 4°C

  • Use tyramide signal amplification for enhanced sensitivity

  • Consider tissue-specific fixation protocols as described in section 2.2

• Inconsistent results between experiments:

  • Standardize tissue processing (time from collection to fixation)

  • Use consistent lot numbers of antibodies

  • Include internal controls in each experiment

  • Document detailed protocols including minor technical adjustments

• Cross-reactivity concerns:

  • Validate with PLEKHA7 knockdown controls

  • Pre-absorb antibodies with recombinant PLEKHA7 fragments

  • Use highly purified antibodies (>95% protein G purified)

  • Perform careful negative controls in tissues known to lack PLEKHA7 expression

How should researchers interpret contradictory results between PLEKHA7 mRNA expression and protein detection in heart tissue?

When confronted with the contradiction between PLEKHA7 mRNA and protein detection in heart tissue, researchers should consider several methodological and biological explanations:

• Epitope accessibility issues: The apparent contradiction between northern blot detection of PLEKHA7 mRNA in heart tissue and the lack of immunofluorescent labeling in heart intercalated disks may reflect tissue-specific epitope modifications. Researchers should attempt alternative fixation protocols (acetone vs. PFA), different antigen retrieval methods, or antibodies targeting different PLEKHA7 epitopes.

• Post-transcriptional regulation: Heart tissue may express PLEKHA7 mRNA but regulate protein synthesis post-transcriptionally. Researchers should quantify both mRNA (by qRT-PCR) and protein levels (by Western blot) from the same tissue samples to confirm this discrepancy.

• Protein degradation: Heart-specific proteases might rapidly degrade PLEKHA7, allowing detection of mRNA but not protein. Researchers should collect fresh tissue samples and use enhanced protease inhibitor cocktails during protein extraction.

• Alternative splicing: Heart tissue may express PLEKHA7 splice variants that lack the epitopes recognized by standard antibodies. Researchers should use antibodies targeting different regions of PLEKHA7 and perform RT-PCR with primers designed to detect potential heart-specific isoforms.

• Methodological approach comparison: To resolve this discrepancy, researchers should compare results from multiple detection methods:

What methodological considerations should be implemented when comparing PLEKHA7 expression between normal and pathological tissues?

When comparing PLEKHA7 expression between normal and pathological tissues, researchers should implement these methodological considerations to ensure valid comparisons:

• Standardized tissue collection and processing:

  • Match collection protocols between normal and pathological samples

  • Control for post-mortem interval in cadaveric tissues

  • Standardize fixation time and conditions

  • Process control and experimental samples in parallel

• Quantitative expression analysis:

  • Use multiple techniques (qRT-PCR, Western blot, immunohistochemistry) to confirm findings

  • Employ digital image analysis for quantifying immunohistochemical staining intensity

  • Include loading controls and housekeeping genes appropriate for the pathology being studied

  • Normalize expression to tissue-specific reference genes that remain stable in the pathological condition

• Genotype-phenotype correlations:

  • Consider PLEKHA7 genotype at SNP rs11024102, as the C risk allele correlates with reduced PLEKHA7 expression in PACG

  • Stratify samples by genotype to avoid confounding expression differences

  • Control for demographic factors that might influence expression independent of pathology

• Controls for epitope accessibility:

  • Include positive control tissues known to express PLEKHA7

  • Use multiple antibodies targeting different PLEKHA7 epitopes

  • Employ antigen retrieval optimization for each tissue type

  • Consider that pathological tissues may have altered protein crosslinking affecting epitope recognition

• Function-expression correlation:

  • Correlate PLEKHA7 expression changes with functional outcomes (e.g., junction integrity, barrier function)

  • Assess downstream effects on Rac1/Cdc42 activity when PLEKHA7 expression is altered

  • Evaluate expression patterns of interacting proteins simultaneously

• Technical validation:

  • Confirm antibody specificity in both normal and pathological contexts using appropriate controls

  • Include gradient standards of recombinant PLEKHA7 for quantitative Western blot analysis

  • Use automated staining platforms to minimize batch effects in immunohistochemistry

By implementing these methodological considerations, researchers can generate reliable comparative data on PLEKHA7 expression between normal and pathological tissues, particularly in PACG and other conditions where PLEKHA7 dysregulation may play a pathogenic role.

How might PLEKHA7 antibodies be utilized to explore its potential role in cancer development and progression?

PLEKHA7 antibodies can be instrumental in investigating its potential role in cancer through several research approaches:

• Tumor tissue microarray analysis: PLEKHA7 antibodies can be used to quantify expression across large numbers of tumor samples and matched normal tissues. This approach would test the hypothesis suggested in previous research that "AJ stabilization through PLEKHA7-dependent microtubule anchoring is important in cancer development and progression" .

• Junction stability assessment in tumor progression: Using PLEKHA7 antibodies in combination with other adherens junction markers, researchers can track changes in junction integrity during epithelial-to-mesenchymal transition (EMT). Since PLEKHA7 has been proposed to regulate AJ stability through its ability to link the microtubule cytoskeleton to E-cadherin , its dysregulation may contribute to junction breakdown during tumor progression.

• Correlation with metastatic potential: Researchers can compare PLEKHA7 expression and localization between primary tumors and their metastases using immunohistochemistry with PLEKHA7 antibodies. This could reveal whether PLEKHA7 downregulation correlates with increased metastatic potential.

• Functional studies in cancer cell lines: After manipulating PLEKHA7 expression in cancer cell lines, researchers can use antibodies to confirm knockdown or overexpression while assessing effects on:

  • Cell migration and invasion capabilities

  • Rac1/Cdc42 activity and actin cytoskeleton organization

  • Formation and stability of cell-cell contacts

• Tumor histological typing: Given that "specific antibodies against PLEKHA7 could be useful in the histological typing and diagnosis of tumors" , researchers can develop PLEKHA7 immunohistochemistry panels alongside other junction proteins to improve tumor classification, particularly in carcinomas of epithelial origin.

What novel methodological approaches could researchers develop to study the interaction between PLEKHA7 and the microtubule cytoskeleton?

Researchers could develop several novel methodological approaches to study PLEKHA7-microtubule interactions:

• Live-cell super-resolution microscopy: Combining PLEKHA7 antibody fragments (Fab) conjugated to photoswitchable fluorophores with tubulin markers could enable super-resolution imaging of dynamic interactions between PLEKHA7 and microtubules in living cells. This would provide nanoscale spatial resolution (~20nm) of these interactions at adherens junctions.

• Proximity ligation assays (PLA): Using PLEKHA7 antibodies in combination with anti-tubulin antibodies in PLA workflows would generate fluorescent signals only when PLEKHA7 and microtubule proteins are within 40nm of each other. This approach could map regions of interaction across different cell types and experimental conditions.

• FRET-based interaction sensors: Developing FRET biosensors using PLEKHA7 antibody-based detection coupled with fluorescently-tagged tubulin would allow real-time monitoring of interaction dynamics during junction assembly, disassembly, and cellular responses to mechanical stress.

• Domain-specific interaction mapping: Using antibodies against specific PLEKHA7 domains (WW domains, PH domain, coiled-coil domains, proline-rich domains) in pull-down assays with microtubule components would identify which domains are critical for microtubule interactions.

• Correlative light-electron microscopy: Combining immunofluorescence using PLEKHA7 antibodies with electron microscopy would provide ultrastructural details of how PLEKHA7 bridges adherens junctions and microtubules, expanding on previous immunoelectron microscopy findings that localized PLEKHA7 at a mean distance of 28 nm from the plasma membrane .

• Microtubule plus-end tracking: Using PLEKHA7 antibodies alongside markers for microtubule plus-end tracking proteins (+TIPs) would reveal whether PLEKHA7 interacts preferentially with growing microtubule ends as they approach adherens junctions.

How can PLEKHA7 antibody-based research advance our understanding of epithelial barrier function in health and disease?

PLEKHA7 antibody-based research can significantly advance our understanding of epithelial barrier function through several innovative approaches:

• Barrier development and maturation studies: Using PLEKHA7 antibodies to track its expression and localization during epithelial differentiation could reveal critical checkpoints in barrier establishment. The observation that PLEKHA7 shows a "modulated gradient of expression that correlates with cell differentiation" in intestinal epithelia provides a foundation for such studies.

• Mechanistic investigation of BAB dysfunction in PACG: Since PLEKHA7 has been identified as a regulator of blood-aqueous barrier function and is downregulated in PACG patients , antibody-based studies could elucidate how this downregulation affects barrier integrity through its Rac1/Cdc42 GAP activity.

• Cross-barrier comparative analysis: Using PLEKHA7 antibodies to compare its role across different barrier tissues (intestinal, renal, ocular, blood-brain barrier) could reveal tissue-specific mechanisms of barrier regulation. This is supported by findings showing tissue-specific distribution patterns of PLEKHA7 .

• Junction-cytoskeleton coordination in barrier restoration: Following barrier disruption (by calcium depletion, inflammatory mediators, or mechanical stress), PLEKHA7 antibodies could track its recruitment during junction reassembly, providing insights into the temporal coordination between adherens junction formation and cytoskeletal reorganization.

• Disease-specific barrier dysfunction: In conditions with known barrier defects (inflammatory bowel disease, diabetic retinopathy, chronic kidney disease), PLEKHA7 antibody studies could determine whether altered PLEKHA7 expression or localization contributes to pathogenesis through its effects on the paracellular barrier between epithelial cells .

• Therapeutic target validation: As potential barrier-enhancing therapeutics are developed, PLEKHA7 antibodies could serve as tools to validate target engagement and monitor changes in PLEKHA7 expression, localization, and downstream signaling events affecting Rac1/Cdc42 activity.

By applying these approaches, researchers can build a comprehensive understanding of how PLEKHA7 contributes to epithelial barrier function in both physiological and pathological states, potentially leading to new therapeutic strategies for barrier dysfunction disorders.