PCDH7 Antibody

What is PCDH7 and what cellular functions does it perform?

Protocadherin-7 (PCDH7) is a member of the protocadherin family within the cadherin superfamily. In humans, the canonical protein has 1069 amino acid residues with a molecular mass of 116.1 kDa and is localized to the cell membrane . PCDH7 is highly expressed in the brain and heart, with lower levels in various other tissues . Functionally, PCDH7 is involved in:

• Cell-cell adhesion processes

• Inhibition of homotypic cell-in-cell structure formation

• Regulation of actomyosin contraction at cell-cell contacts

• Interaction with protein phosphatase 1 (PP1α) to influence phosphorylation of myosin light chain 2 (pMLC2)

Alternative names include BHPCDH, PPP1R120, BH-protocadherin (brain-heart), and brain-heart protocadherin .

What applications are PCDH7 antibodies commonly used for?

PCDH7 antibodies are utilized across multiple experimental platforms in research settings:

The versatility of these antibodies makes them valuable for investigating PCDH7 expression, localization, and molecular interactions in different experimental contexts .

What should researchers consider when selecting a PCDH7 antibody?

When selecting a PCDH7 antibody for research, consider the following factors:

• Antibody Type: Mouse monoclonal (e.g., clone OTI2G6) versus rabbit polyclonal antibodies offer different specificity and sensitivity profiles

• Target Region: C-terminal versus other epitopes may detect different isoforms due to alternative splicing (PCDH7 exists in multiple isoforms)

• Species Reactivity: Confirm cross-reactivity with your species of interest (human, mouse, rat, etc.)

• Validated Applications: Ensure the antibody has been validated for your specific application (WB, IHC, IF, etc.)

• Clone Considerations: Different clones may have different performance characteristics (e.g., OTI2G6 clone has been cited in multiple publications)

For studies involving specific isoforms, carefully review the antibody's immunogen sequence to ensure it will detect your isoform of interest .

What are the optimal conditions for detecting PCDH7 using Western blot?

Optimizing Western blot conditions for PCDH7 detection requires attention to several technical parameters:

• Sample Preparation: Total cell lysates from HEK293T, HeLa, Jurkat, TCMK1, or NIH 3T3 have been successfully used

• Protein Amount: Load approximately 50 μg of total protein per lane

• Antibody Dilution: Starting dilutions of 1:200 to 1:2000 are recommended, with optimization required for each specific antibody

• Expected Band Size: The predicted molecular weight is 116 kDa

• Denaturation Conditions: Note that for some applications, such as detecting anti-PCDH7 antibodies in patient sera, non-reduced conditions may be preferable

• Detection Method: ECL (enhanced chemiluminescence) technique has been successfully used with exposure times around 3 minutes

When troubleshooting, consider that heavily glycosylated regions of PCDH7 (particularly amino acids 689-845) may affect band appearance or intensity .

How should PCDH7 antibodies be used for immunohistochemistry and immunofluorescence?

For successful IHC and IF detection of PCDH7, researchers should follow these protocol guidelines:

For IHC:

• Tissue Preparation: 5 μm FFPE tissue sections are suitable

• Epitope Retrieval: Use high-pH EDTA-based retrieval solution (e.g., Epitope Retrieval 2 from Leica) for 20 minutes

• Antibody Dilution: Starting dilution of 1:400 in background reducing diluent with 15-minute incubation

• Detection System: Polymer Refine Detection System or equivalent with DAB visualization (10-minute incubation)

• Counterstaining: Schmidt hematoxylin (5 minutes) provides good nuclear contrast

For IF:

• Retrieval: High-pH target retrieval solution in pressure cooker for 30 minutes

• Primary Antibody: Dilution of 1:200 in blocking solution (2% calf fetal serum and 2% normal goat serum) with overnight incubation at 4°C

• Secondary Antibody: Goat Alexa 488-conjugated Fab IgG anti-mouse antibodies at 1:400 dilution

• For Co-localization: Additional antibodies (e.g., anti-human IgG Alexa Fluor 647) can be used at 1:50 dilution

• Mounting: Use appropriate mounting medium compatible with fluorescence imaging

These protocols have been successfully used to demonstrate PCDH7 localization, particularly at the glomerular basement membrane in kidney tissue studies .

How can PCDH7 antibodies be used to study membranous nephropathy?

PCDH7 has been identified as a novel glomerular autoantigen in a subset of patients with PLA2R-negative membranous nephropathy (MN) . Researchers studying this condition can:

• Detect PCDH7 Autoantigen: Use immunohistochemistry to visualize granular staining along the glomerular basement membrane (GBM) in kidney biopsies

• Identify PCDH7-Associated MN: Screen for PCDH7 positivity in patients negative for other known MN antigens (PLA2R, THSD7A, EXT1/EXT2, NELL1, and SEMA3B)

• Detect Anti-PCDH7 Antibodies: Perform Western blot analysis using patient sera against recombinant PCDH7 under non-reduced conditions

• Elution Studies: Extract and detect anti-PCDH7 IgG from kidney biopsy tissue

• Co-localization Analysis: Use confocal microscopy to demonstrate co-localization of PCDH7 and IgG along the GBM

The identification of PCDH7 as a target antigen opens new avenues for diagnosing and potentially treating this specific subtype of membranous nephropathy, which represents approximately 5.7% of PLA2R-negative MN cases .

What technical challenges exist in detecting PCDH7 by mass spectrometry?

Mass spectrometry detection of PCDH7 presents unique challenges that researchers should be aware of:

• Low Spectral Counts: PCDH7 typically shows lower spectral counts in MS/MS compared to other antigens like PLA2R, EXT1/EXT2, or NELL1

• Glycosylation Interference: PCDH7 is heavily glycosylated, particularly in amino acids 689-845, which can interfere with trypsin's access and inhibit binding/cleavage of arginine and lysine residues

• Fragmentation Challenges: During higher-energy collisional dissociated MS/MS fragmentation, glycan chains are preferentially fragmented at the expense of peptide bonds, resulting in spectra dominated by glycan fragments rather than amino acid spectra

• Sequence Coverage Gaps: Examination of representative sequence coverage reveals that the most heavily glycated regions (amino acids 689-845) are often not represented in the coverage sequence

Despite these challenges, PCDH7 can be reliably detected by MS/MS if unique peptides are identified, even with low spectral counts . This approach has successfully identified PCDH7 in glomeruli of PLA2R-negative MN cases.

How can researchers study PCDH7's role in inhibiting homotypic cell-in-cell structure formation?

PCDH7 has been identified as an inhibitor of homotypic cell-in-cell (hoCIC) structure formation . Researchers interested in this function can:

• Manipulate PCDH7 Expression: Use overexpression or siRNA knockdown approaches to modulate PCDH7 levels

• siRNA Sequences: Use validated siRNA sequences for PCDH7 depletion:

  • 1#: sense-5′-CCAAGCUAUGAAAUUAGCAAATT-3′, antisense-5′-UUUGCUAAUUUCAUAGCUUGGTT-3′

  • 2#: sense-5′-GCUGGCAUUAUGACGGUGAUUTT-3′, antisense-5′-AAUCACCGUCAUAAUGCCAGCTT-3′

• Assess Cell Stiffness: Use agarose compression assays to measure relative stiffness of cells expressing or depleted of PCDH7

• Analyze pMLC2 Levels: Monitor phosphorylated myosin light chain 2 (pMLC2) levels as a readout of actomyosin contraction, particularly at cell-cell contacts

• Study PCDH7-PP1α Interaction: Perform co-immunoprecipitation to detect interaction between PCDH7 and protein phosphatase 1α (PP1α)

• Evaluate PP1α Knockdown: Use siRNA to deplete PP1α and observe effects on pMLC2 levels and CIC formation

This research approach has revealed that PCDH7 inhibits hoCIC formation by interacting with and inactivating PP1α, thereby increasing pMLC2 levels and enhancing actomyosin contraction at cell-cell contacts .

How can researchers validate the specificity of PCDH7 antibodies?

Validating antibody specificity is crucial for reliable PCDH7 research. Recommended validation strategies include:

• Positive Controls: Use cell lines known to express PCDH7, such as HEK293T, HeLa, Jurkat, TCMK1, and NIH 3T3

• Recombinant Expression: Test antibody against cells transfected with PCDH7 expression constructs versus empty vector controls

• Multiple Antibodies: Use antibodies from different sources or targeting different epitopes and compare the results

• Knockdown/Knockout Validation: Perform siRNA knockdown of PCDH7 and confirm reduced signal with the antibody

• Cross-Reactivity Testing: Test specificity against multiple species when conducting comparative studies

• Blocking Peptides: Use specific blocking peptides (corresponding to the immunogen) to confirm signal specificity

For studies involving specific PCDH7 isoforms, validate whether the antibody recognizes all or only specific isoforms by using recombinant proteins of different isoforms .

What are common pitfalls when working with PCDH7 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with PCDH7 antibodies:

• Glycosylation Effects: Heavy glycosylation of PCDH7 may affect antibody binding, particularly for antibodies targeting glycosylated regions (amino acids 689-845)

  • Solution: Use antibodies targeting non-glycosylated regions or treat samples with glycosidases

• Isoform Specificity: Alternative splicing produces multiple PCDH7 isoforms that may not all be recognized by a given antibody

  • Solution: Verify which isoforms your antibody detects and select one that recognizes your isoform of interest

• Subcellular Localization Variability: PCDH7 localizes primarily at cell-cell contacts but may have different patterns in various cell types

  • Solution: Include appropriate positive controls specific to your cell type

• Low Expression Levels: In some tissues, PCDH7 may be expressed at low levels, making detection challenging

  • Solution: Optimize protein loading amounts and use more sensitive detection methods

• Cross-Reactivity: Some antibodies may cross-react with other protocadherins due to sequence homology

  • Solution: Perform appropriate controls and consider using monoclonal antibodies for higher specificity

Addressing these pitfalls requires careful experimental design and thorough validation to ensure reliable and reproducible results in PCDH7 research.

How are PCDH7 antibodies being used to explore novel disease associations?

Recent research has revealed important disease associations for PCDH7, with antibodies playing a crucial role in these discoveries:

• Membranous Nephropathy: PCDH7 has been identified as a novel glomerular autoantigen in approximately 5.7% of PLA2R-negative membranous nephropathy cases

  • Using immunohistochemistry, researchers detected granular PCDH7 staining along the glomerular basement membrane

  • Anti-PCDH7 antibodies were detected in both serum and kidney biopsy tissue from these patients

• Cancer Research: PCDH7 expression changes have been associated with various cancers

  • PCDH7 enhances anchorage-independent cell growth in a hoCIC-dependent manner

  • Antibodies are being used to study PCDH7's role in tumor progression

• Cell Biology: PCDH7 has been shown to inhibit homotypic cell-in-cell structure formation

  • Antibodies help visualize PCDH7 localization at cell-cell contacts

  • Immunoprecipitation experiments with PCDH7 antibodies revealed interaction with PP1α

These emerging applications highlight the importance of specific and well-validated PCDH7 antibodies in expanding our understanding of PCDH7's role in health and disease.

What methodological advances are improving PCDH7 detection and characterization?

Recent technological and methodological advances have enhanced our ability to detect and characterize PCDH7:

• Mass Spectrometry Approaches: Despite challenges with glycosylated proteins, laser microdissection and mass spectrometry (MS/MS) have successfully identified PCDH7 in glomeruli of membranous nephropathy patients

  • Improved protocols account for glycosylation interference

  • Even low spectral counts can provide reliable identification

• Confocal Microscopy: Advanced confocal techniques allow precise co-localization studies, such as demonstrating PCDH7 and IgG co-localization along the glomerular basement membrane

• Elution Studies: Techniques to elute IgG from frozen tissue samples have enabled detection of anti-PCDH7 antibodies in kidney biopsy material

• Recombinant Protein Technologies: Production of specific PCDH7 isoforms as recombinant proteins facilitates more precise antibody validation and functional studies

• Functional Assays: Development of specialized assays for cell stiffness measurement (agarose compression assay) has allowed researchers to connect PCDH7 function to cellular mechanics

These advances provide researchers with more sophisticated tools to study PCDH7 expression, localization, interactions, and function in various biological and pathological contexts.