PLEKHA7 Antibody, Biotin conjugated

What is PLEKHA7 and why is it important in cell biology research?

PLEKHA7 (Pleckstrin Homology Domain Containing Family A Member 7) is a cytoplasmic component of the epithelial adherens junction belt that links the E-cadherin-p120 catenin complex to the microtubule cytoskeleton. It contains two WW domains and one pleckstrin homology (PH) domain in its N-terminal region, plus coiled-coil and proline-rich domains in its C-terminal half . PLEKHA7 plays a critical role in maintaining epithelial tissue integrity through its specific localization at adherens junctions. Unlike other adherens junction proteins, PLEKHA7 displays a unique subcellular distribution pattern, being concentrated at the apical junctional belt but absent from the lateral regions of polarized epithelial cells . This distinct localization pattern makes PLEKHA7 a valuable marker for studying specialized junctional complexes in epithelial tissues.

What is the tissue distribution pattern of PLEKHA7 protein?

PLEKHA7 exhibits a specific tissue distribution pattern primarily in epithelial tissues. Immunofluorescence studies have demonstrated PLEKHA7 presence at epithelial junctions in kidney, liver, pancreas, intestine, retina, and cornea . In kidney cortex, PLEKHA7 is detected in convoluted tubules but notably absent from glomeruli . In pancreatic tissue, PLEKHA7 localizes to epithelial cells of acini and ductules but is not detected in islets of Langerhans . Liver sections show PLEKHA7 labeling along junctions between epithelial cells lining bile canaliculi . In intestinal tissues (duodenum, colon, ileum), PLEKHA7 appears as a thin apical bar in longitudinal sections and as a net-like meshwork in tangential sections of enterocytes . In the retina, PLEKHA7 is detected in the outer limiting membrane and along junctions between retinal pigmented epithelial cells . Interestingly, PLEKHA7 is not clearly detectable in endothelial junctions of blood vessels or in intercalated disks of cardiac tissue, despite northern and western blot analyses confirming PLEKHA7 mRNA and protein expression in heart tissue .

What are the optimal fixation and permeabilization protocols for immunolocalization studies using biotin-conjugated PLEKHA7 antibodies?

For optimal immunolocalization of PLEKHA7 using biotin-conjugated antibodies, tissue preparation and fixation protocols must preserve epitope accessibility while maintaining cellular architecture. Paraformaldehyde fixation (4%) for 15-20 minutes at room temperature followed by permeabilization with 0.2% Triton X-100 has proven effective for most epithelial tissues . Methanol fixation (-20°C for 5 minutes) represents an alternative that combines fixation and permeabilization in one step and may better preserve certain PLEKHA7 epitopes. For frozen tissue sections, it's critical to use freshly prepared 4% paraformaldehyde and limit fixation time to minimize epitope masking.

When working with biotin-conjugated antibodies specifically, consider that some tissues (particularly liver and kidney) contain endogenous biotin that may produce background signal. Pre-blocking with avidin followed by biotin (avidin-biotin blocking kit) before antibody incubation can significantly reduce this background. Additionally, using TBS rather than PBS buffers may improve signal-to-noise ratio when working with biotin-streptavidin detection systems in PLEKHA7 localization studies.

How can I validate the specificity of a biotin-conjugated PLEKHA7 antibody?

Validating antibody specificity is crucial for reliable PLEKHA7 research. A comprehensive validation approach should include several complementary techniques:

• Western blot analysis: Confirm detection of the expected 135-145 kDa bands in epithelial tissues and cells, with reduced signal in PLEKHA7-depleted cells through shRNA-mediated silencing .

• Immunofluorescence with knockdown controls: Perform immunostaining on cells where PLEKHA7 has been depleted through siRNA or shRNA approaches, which should show significantly reduced labeling compared to control cells .

• Peptide competition assay: Pre-incubate the biotin-conjugated antibody with excess immunizing peptide (if available) before application to western blots or tissue sections. Specific signals should be significantly reduced or eliminated.

• Comparison with alternative antibodies: Compare staining patterns with other validated PLEKHA7 antibodies targeting different epitopes.

• Co-localization studies: Confirm that the biotin-conjugated PLEKHA7 antibody co-localizes with known adherens junction markers (E-cadherin, p120 catenin) at the apical junctional belt but not along lateral membranes in polarized epithelial cells .

What detection systems work best with biotin-conjugated PLEKHA7 antibodies for immunofluorescence microscopy?

Biotin-conjugated PLEKHA7 antibodies offer flexibility in detection strategies for immunofluorescence microscopy. The following detection systems have proven effective, each with distinct advantages:

• Streptavidin-fluorophore conjugates: Using streptavidin conjugated to fluorophores (Alexa Fluor 488, 555, 647) provides direct detection with high sensitivity. Select fluorophores that complement other markers in multi-labeling experiments. The high affinity of streptavidin for biotin (Kd ≈ 10^-15 M) ensures stable binding and low background.

• Tyramide signal amplification (TSA): For tissues with low PLEKHA7 expression, implementing TSA with streptavidin-HRP followed by fluorophore-conjugated tyramide can amplify signal 50-100 fold over conventional methods. This approach is particularly valuable when examining PLEKHA7 in tissues like retina or cornea where expression may be lower.

• Quantum dot-streptavidin conjugates: These provide exceptional photostability for long-term imaging or repeated scanning, with narrow emission spectra facilitating multi-labeling studies of junctional complexes containing PLEKHA7.

When selecting a detection system, consider that PLEKHA7 localizes approximately 28 nm from the plasma membrane at adherens junctions , so detection systems with minimal spatial displacement (direct fluorophore conjugates) may provide more precise localization information compared to multilayer detection systems.

How does PLEKHA7 subcellular localization differ from other adherens junction proteins, and what implications does this have for experimental design?

This unique localization pattern has several important implications for experimental design:

• Control selection: When designing co-localization experiments, researchers should include both markers that fully overlap with PLEKHA7 (e.g., apical junction-specific proteins) and those that show partial overlap (e.g., E-cadherin) to accurately characterize junctional distributions.

• Tissue selection: Given the absence of PLEKHA7 from certain structures like kidney glomeruli or heart intercalated disks where other junction proteins are present, careful tissue selection is crucial for comparative studies.

• Resolution requirements: The precise localization of PLEKHA7 approximately 28 nm from the plasma membrane necessitates high-resolution imaging techniques (super-resolution microscopy, immuno-EM) for detailed localization studies.

• Junction disruption studies: Experimental approaches that selectively disrupt different junctional components may reveal PLEKHA7's specific role in maintaining apical junction integrity distinct from lateral adhesions.

What approaches can be used to study PLEKHA7 isoforms in different tissues?

PLEKHA7 exists in multiple isoforms, with northern blotting revealing two major mRNA transcripts (~5.5 kb and ~6.5 kb) and immunoblotting identifying major polypeptides of ~135 kDa and ~145 kDa . To effectively study these isoforms using PLEKHA7 antibodies, consider the following approaches:

• Isoform-specific antibody selection: If the biotin-conjugated PLEKHA7 antibody targets a region common to all isoforms, complement with isoform-specific antibodies targeting unique regions. Verify epitope locations relative to known isoform sequence differences before selecting antibodies.

• Two-dimensional gel electrophoresis: Combine isoelectric focusing with SDS-PAGE followed by immunoblotting with biotin-conjugated PLEKHA7 antibodies to distinguish isoforms that may have similar molecular weights but different post-translational modifications.

• RT-PCR with isoform-specific primers: Correlate protein detection patterns with mRNA isoform expression through parallel analysis of tissue samples.

• Immunoprecipitation coupled with mass spectrometry: Use biotin-conjugated PLEKHA7 antibodies for immunoprecipitation followed by mass spectrometric analysis to identify specific isoforms and potential post-translational modifications.

• Tissue comparative analysis: Systematically compare PLEKHA7 detection patterns across tissues known to express different isoform ratios (based on the northern blot data showing tissue-specific expression patterns of the 5.5 kb versus 6.5 kb transcripts) .

How can I optimize biotin-conjugated PLEKHA7 antibodies for use in super-resolution microscopy?

Super-resolution microscopy techniques can reveal detailed information about PLEKHA7's precise localization at adherens junctions, particularly given its specific distance (approximately 28 nm) from the plasma membrane . Optimizing biotin-conjugated PLEKHA7 antibodies for these applications requires special considerations:

• Fixation optimization: Standard paraformaldehyde fixation may cause epitope masking or protein crosslinking that limits resolution. Test glyoxal-based fixatives or combinations of light paraformaldehyde (2%) with glutaraldehyde (0.05-0.1%) to improve ultrastructural preservation while maintaining antibody accessibility.

• Signal density control: For techniques like STORM or PALM, adjust biotin-conjugated antibody concentration to achieve optimal fluorophore density (typically 500-2000 fluorophores per μm²). Too high density causes signal overlap, while too low density provides insufficient sampling.

• Secondary label selection: For STED microscopy, use streptavidin conjugated to photostable dyes with appropriate depletion wavelengths (e.g., STAR 635P, ATTO 647N). For STORM/PALM, select streptavidin conjugated to photoswitchable fluorophores (Alexa Fluor 647, Cy5).

• Multi-color optimization: When studying PLEKHA7 relative to other junctional proteins, carefully select fluorophore combinations that minimize channel crosstalk and have compatible photophysical properties for the chosen super-resolution technique.

• Sample mounting media: Use oxygen-scavenging systems (e.g., glucose oxidase/catalase with glucose) for STORM imaging to improve fluorophore photoswitching and extend acquisition time.

What are common causes of false-negative results when using PLEKHA7 antibodies, and how can they be addressed?

False-negative results can significantly impact research findings when working with PLEKHA7 antibodies. Several tissue and methodology-specific factors may contribute to these issues:

• Epitope masking: PLEKHA7's complex domain structure and protein interactions may shield antibody epitopes. Try multiple antigen retrieval methods (heat-induced in citrate buffer pH 6.0, Tris-EDTA pH 9.0, or enzymatic retrieval with proteinase K) to expose masked epitopes.

• Tissue-specific epitope modifications: The PLEKHA7 protein may undergo tissue-specific post-translational modifications. This appears particularly relevant in heart tissue, where despite detection of PLEKHA7 mRNA and protein by northern and western blotting, immunofluorescence labeling of intercalated disks is negative . Try antibodies targeting different PLEKHA7 epitopes when working with specific tissues.

• Fixation-induced epitope destruction: Some epitopes are particularly sensitive to overfixation. Implement a fixation time-course experiment (5, 10, 15, 20 minutes) to determine optimal fixation duration for your specific tissue.

• Biotin blocking inefficiency: When using biotin-conjugated antibodies, endogenous biotin in tissues like liver and kidney may saturate the detection system. Implement a double sequential blocking protocol with avidin followed by biotin before antibody application.

• Species cross-reactivity limitations: If antibodies were raised against human PLEKHA7, they may show limited reactivity with orthologues from other species. Research results demonstrate that some rabbit antibodies recognize human but not canine PLEKHA7 in immunofluorescence applications despite detecting both in western blots .

How can I minimize background and optimize signal-to-noise ratio when using biotin-conjugated PLEKHA7 antibodies?

Achieving optimal signal-to-noise ratio is essential for accurate PLEKHA7 localization. The following strategies address common sources of background with biotin-conjugated antibodies:

• Endogenous biotin blocking: Pre-block endogenous biotin using an avidin-biotin blocking kit before applying the biotin-conjugated PLEKHA7 antibody. This is particularly important in biotin-rich tissues like liver, kidney, and brain.

• Optimized antibody concentration: Titrate the biotin-conjugated PLEKHA7 antibody to determine the minimum concentration needed for specific signal detection. Excessive antibody concentration increases non-specific binding.

• Sequential multi-labeling: When performing multi-label immunofluorescence with other biotin-conjugated antibodies, complete each detection sequence (biotin-conjugated primary → streptavidin-fluorophore) before beginning the next to prevent cross-reaction.

• Blocking optimization: Use a combination of serum (5-10%) from the species of the detection reagent, plus BSA (1-3%) and 0.1-0.3% Triton X-100 in blocking buffers to minimize both Fc receptor and hydrophobic background binding.

• Tissue autofluorescence reduction: Treat sections with Sudan Black B (0.1% in 70% ethanol) for 5-10 minutes after immunodetection to reduce autofluorescence, particularly in tissues like retina where lipofuscin can generate significant background.

• Negative control inclusion: Always include a negative control where the biotin-conjugated PLEKHA7 antibody is omitted but all other detection reagents are applied to identify background from the detection system itself.

What are effective protocols for studying PLEKHA7 interactions with other junctional proteins using biotin-conjugated antibodies?

Investigating PLEKHA7's interactions with other junctional proteins reveals important insights into adherens junction structure and function. Biotin-conjugated PLEKHA7 antibodies can be particularly valuable in these studies through the following protocols:

• Proximity Ligation Assay (PLA): Combine biotin-conjugated PLEKHA7 antibody with antibodies against potential interaction partners (e.g., p120 catenin, E-cadherin) followed by appropriate PLA probes to visualize protein interactions with nanometer resolution. This technique can confirm the previously identified interaction between PLEKHA7 and the E-cadherin-p120 catenin complex .

• Co-immunoprecipitation with biotin-based pull-down: Use biotin-conjugated PLEKHA7 antibodies with streptavidin-coated beads to pull down PLEKHA7 and associated proteins under native conditions. This approach preserves protein complexes better than traditional IP methods due to the high affinity of the biotin-streptavidin interaction.

• FRET analysis using biotin-conjugated primary antibodies: Combine biotin-conjugated PLEKHA7 antibody (detected with streptavidin-donor fluorophore) and antibodies against potential interaction partners (labeled with acceptor fluorophores) to measure FRET signals indicating close proximity (<10 nm) between proteins.

• Sequential immunofluorescence for co-localization studies: Perform detailed co-localization analysis of PLEKHA7 with other junctional proteins (E-cadherin, p120 catenin, β-catenin, afadin) using high-resolution confocal microscopy. The research data shows PLEKHA7 co-localizes with these proteins at the apical junctional belt but, unlike most of them, is absent from the lateral membrane .

How can biotin-conjugated PLEKHA7 antibodies be used to study junction dynamics in live cellular systems?

While conventional immunofluorescence provides static snapshots of PLEKHA7 localization, studying junction dynamics requires specialized approaches. Biotin-conjugated PLEKHA7 antibodies can be adapted for dynamic studies through several innovative techniques:

• Antibody fragment preparation: Generate Fab or scFv fragments from biotin-conjugated PLEKHA7 antibodies for live-cell applications, as their smaller size improves tissue penetration and reduces interference with protein function.

• Quantum dot labeling for single-particle tracking: Conjugate streptavidin-coated quantum dots to biotin-labeled antibody fragments to track individual PLEKHA7 molecules at adherens junctions over extended periods, revealing dynamic behaviors not visible in fixed samples.

• Microinjection approaches: In specialized cell systems where junctions are accessible (e.g., early embryos), microinject biotin-conjugated PLEKHA7 antibody fragments followed by fluorescent streptavidin to visualize junction remodeling during developmental processes.

• Cell-penetrating peptide conjugation: Modify biotin-conjugated antibody fragments with cell-penetrating peptides to facilitate entry into live cells for dynamic imaging studies.

• Correlative light-electron microscopy: Use biotinylated PLEKHA7 antibodies with gold-conjugated streptavidin for post-fixation visualization of PLEKHA7 dynamics captured in live cells that are subsequently processed for electron microscopy.

What are emerging research directions in PLEKHA7 biology where biotin-conjugated antibodies will be particularly valuable?

Several emerging research areas in PLEKHA7 biology will benefit from the application of biotin-conjugated antibodies:

• PLEKHA7's role in microtubule organization: Given PLEKHA7's established link between adherens junctions and the microtubule cytoskeleton , biotin-conjugated antibodies will be valuable for investigating this connection through super-resolution microscopy and proximity labeling approaches.

• Tissue-specific isoform functions: Research data indicates tissue-specific expression patterns of PLEKHA7 mRNA transcripts . Biotin-conjugated antibodies targeting common or isoform-specific regions will help elucidate functional differences between these variants.

• PLEKHA7 in epithelial-mesenchymal transition (EMT): As a component of epithelial junctions, PLEKHA7 may play roles in EMT processes relevant to development and cancer. Biotin-conjugated antibodies enable multiplexed analysis of PLEKHA7 alongside EMT markers.

• Junction-associated signaling complexes: PLEKHA7's distinct localization at the apical junction belt but not lateral membranes suggests involvement in specialized signaling complexes. Biotinylated antibodies can be used in BioID or APEX proximity labeling approaches to identify novel PLEKHA7 interaction partners.

• 3D tissue organization studies: PLEKHA7's role in epithelial organization can be studied in organoid systems using biotin-conjugated antibodies with clearing techniques and light-sheet microscopy to visualize junction patterns in intact 3D structures.