CDH15 Antibody

What is CDH15 and why is it important in scientific research?

CDH15 (Cadherin-15), also known as M-cadherin, is a calcium-dependent cell adhesion protein crucial in myogenic development. It belongs to the classical cadherin family within the cadherin superfamily. CDH15 is primarily expressed in muscle satellite cells, embryonic myotome cells, and hematopoietic bone marrow stem cells . The protein plays a vital role in muscle development by facilitating homotypic binding in trans, allowing for the identification and subsequent fusion of myoblast precursors, particularly in slow-twitch (red fiber) muscles. This process is accompanied by downregulation of mitochondrial-induced apoptosis . CDH15 antibodies are essential tools for studying muscle development, regeneration, and related pathologies.

What are the structural and functional characteristics of CDH15 protein?

Human CDH15 is synthesized as an 814 amino acid preproprecursor with a mass of approximately 88.9 kDa, though it typically appears at 125-130 kDa in Western blots due to post-translational modifications . Mouse CDH15 is synthesized as a 784 amino acid preproprecursor containing:

• A 21 amino acid signal sequence

• A 38 amino acid propeptide

• A 725 amino acid mature region

The mature protein is expressed as a type I transmembrane glycoprotein with:

• A 546 amino acid extracellular region (aa 60-605) containing five consecutive cadherin domains

• A 159 amino acid cytoplasmic domain (aa 626-784)

Mouse CDH15 shares 88% amino acid sequence identity with human CDH15 and 97% with rat CDH15 over amino acids 22-605 .

What are the validated applications for CDH15 antibodies?

Based on multiple supplier validations, CDH15 antibodies have been successfully applied in:

Note: Optimal dilutions should be determined by each laboratory for each application .

How can I optimize Western blot conditions for CDH15 detection?

For optimal Western blot detection of CDH15:

• Sample preparation:

  • Use appropriate lysis buffers (e.g., RIPA) with protease inhibitors

  • Load 30 μg of protein per lane under reducing conditions

• Gel electrophoresis:

  • Use 5-20% SDS-PAGE gel

  • Run at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours

• Transfer:

  • Transfer to PVDF membrane at 150 mA for 50-90 minutes

  • Use Immunoblot Buffer Group 1 or 8 as appropriate

• Blocking:

  • Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

• Antibody incubation:

  • Primary antibody: 0.5-2 μg/mL overnight at 4°C

  • Wash with TBS-0.1% Tween 3 times (5 minutes each)

  • Secondary antibody: HRP-conjugated anti-species IgG at 1:5000 for 1.5 hours at room temperature

• Detection:

  • Develop using enhanced chemiluminescent (ECL) detection kit

  • Expect a specific band at approximately 125-130 kDa

What are the recommended protocols for immunohistochemistry with CDH15 antibodies?

For paraffin-embedded tissue sections:

• Heat-mediated antigen retrieval:

  • Use EDTA buffer (pH 8.0) for optimal epitope retrieval

• Blocking:

  • Block tissue section with 10% goat serum

• Primary antibody incubation:

  • Incubate with CDH15 antibody (2 μg/ml) overnight at 4°C

• Secondary antibody:

  • Use peroxidase-conjugated anti-species IgG

  • Incubate for 30 minutes at 37°C

• Development:

  • Develop using HRP-conjugated secondary antibody system with DAB as chromogen

For frozen sections or immunofluorescence:

• For fluorescent detection, use appropriate fluorophore-conjugated secondary antibodies (e.g., NorthernLights™ 557-conjugated anti-species IgG)

• Counter-stain with DAPI for nuclear visualization

Why might I observe different molecular weights for CDH15 in Western blots?

The predicted molecular weight of CDH15 is approximately 88.9 kDa, but it typically appears at 125-130 kDa in Western blots . This discrepancy can be attributed to:

• Post-translational modifications, particularly glycosylation

• Different sample preparation conditions (reducing vs. non-reducing)

• Protein-protein interactions that are not fully disrupted

• Differences between species (human vs. mouse vs. rat)

• Splice variants or proteolytic processing

If you observe unexpected bands, consider:

• Validating with positive control lysates (e.g., C2C12 mouse myoblast, L6 rat myoblast, or HeLa cells)

• Using reducing conditions consistently

• Comparing with literature-reported molecular weights

What are common sources of non-specific staining with CDH15 antibodies and how can they be minimized?

Common sources of non-specific staining include:

• Insufficient blocking:

  • Increase blocking time or use alternative blocking agents (BSA, serum)

  • Consider using commercial blocking solutions specifically designed for immunodetection

• Cross-reactivity:

  • Use antibodies verified for specificity (e.g., antigen affinity-purified)

  • Include appropriate negative controls in your experiments

• Excessive antibody concentration:

  • Titrate antibodies to determine optimal concentration

  • Follow manufacturer's recommended dilutions

• Background in immunohistochemistry:

  • Optimize antigen retrieval methods

  • Use appropriate peroxidase blocking steps if using HRP-based detection

  • Consider using tyramide signal amplification methods for weak signals

• Autofluorescence in fluorescent applications:

  • Include an autofluorescence quenching step

  • Use spectral unmixing during image acquisition

How can I validate the specificity of a CDH15 antibody?

To validate CDH15 antibody specificity:

• Positive controls:

  • Use cell lines with known CDH15 expression (C2C12 mouse myoblast, L6 rat myoblast, HeLa)

  • Include tissue positive controls (skeletal muscle, brain cerebellum)

• Negative controls:

  • Omit primary antibody

  • Use isotype control antibodies

  • Use tissues/cells known to lack CDH15 expression

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide before application

  • Signal should be specifically reduced or eliminated

• Genetic validation:

  • Use CDH15 knockout or knockdown models

  • Compare with wild-type expression patterns

• Orthogonal methods:

  • Confirm findings with multiple antibodies targeting different epitopes

  • Validate using complementary techniques (e.g., mRNA expression)

How can CDH15 antibodies be used to study myogenesis and muscle regeneration?

CDH15 antibodies are valuable tools for investigating myogenesis and muscle regeneration:

• Satellite cell identification and activation:

  • CDH15 is expressed in muscle satellite cells and can be used as a marker for their identification

  • Track satellite cell proliferation and differentiation during regeneration using CDH15 immunostaining

• Developmental studies:

  • Examine CDH15 expression patterns during embryonic muscle development

  • Study the transition from myoblasts to mature muscle fibers

• Injury models:

  • Monitor CDH15 expression changes in response to muscle injury

  • Track the temporal dynamics of satellite cell activation and fusion

• Co-localization studies:

  • Combine CDH15 antibodies with other myogenic markers (Pax7, MyoD, Myogenin) to study differentiation stages

  • Use multiplexed immunofluorescence to visualize cellular interactions

• Functional studies:

  • Apply CDH15 antibodies in neutralization experiments to assess functional roles in myoblast fusion

  • Use in calcium-dependent adhesion assays to study homophilic binding properties

What considerations are important when selecting CDH15 antibodies for cross-species experiments?

When selecting CDH15 antibodies for cross-species experiments:

• Sequence homology:

  • Consider the degree of sequence homology between species (mouse CDH15 shares 88% amino acid identity with human and 97% with rat over aa 22-605)

  • Select antibodies raised against conserved epitopes when possible

• Validated reactivity:

  • Choose antibodies explicitly validated in your species of interest

  • Several antibodies are validated for multiple species (human, mouse, rat)

• Application-specific validation:

  • An antibody working in WB may not work in IHC or ICC for all species

  • Request application-specific validation data for your species

• Epitope location:

  • Consider antibodies targeting different domains (extracellular vs. cytoplasmic)

  • Extracellular domain-targeting antibodies may have broader cross-reactivity

• Controls:

  • Include species-specific positive and negative controls

  • Consider using recombinant proteins for standardization

How can post-translational modifications of CDH15 be studied using antibodies?

Studying CDH15 post-translational modifications requires specialized approaches:

• Modification-specific antibodies:

  • Use antibodies specifically recognizing glycosylated, phosphorylated, or otherwise modified CDH15

  • Complement with general CDH15 antibodies to determine modification ratios

• Enzymatic treatments:

  • Treat samples with deglycosylation enzymes (PNGase F, Endo H) before immunodetection

  • Compare migration patterns before and after treatment to assess glycosylation status

• 2D gel electrophoresis:

  • Separate CDH15 based on both isoelectric point and molecular weight

  • Identify post-translational modification patterns using subsequent immunoblotting

• Mass spectrometry:

  • Immunoprecipitate CDH15 using validated antibodies

  • Analyze purified protein by mass spectrometry to identify modification sites

• Subcellular localization:

  • Study how modifications affect localization using immunofluorescence

  • Compare membrane vs. cytoplasmic distribution under different conditions

What are the considerations for using CDH15 antibodies in studies of neurological disorders?

CDH15 has been implicated in neurological disorders, particularly intellectual disability (MRD3) . When studying these conditions:

• Brain region specificity:

  • CDH15 is expressed in the brain and cerebellum

  • Use region-specific analysis to correlate expression with pathology

• Developmental timing:

  • Consider developmental expression patterns of CDH15

  • Select appropriate developmental time points for analysis

• Genetic models:

  • Study CDH15 mutations associated with intellectual disability

  • Compare protein expression and localization between wild-type and mutant models

• Human samples:

  • Validate antibodies specifically for human brain tissue applications

  • Consider post-mortem changes that might affect epitope recognition

• Co-expression analysis:

  • Combine with other neuronal or glial markers for cell type-specific analysis

  • Investigate potential interactions with other neurodevelopmental proteins

How can CDH15 antibodies be integrated into high-throughput or multiplexed platforms?

CDH15 antibodies can be incorporated into advanced research platforms:

• Multiplexed immunofluorescence:

  • Use spectrally distinct fluorophores for co-detection of CDH15 with other markers

  • Implement multispectral imaging for separating overlapping signals

• Mass cytometry (CyTOF):

  • Conjugate CDH15 antibodies with rare earth metals for high-dimensional analysis

  • Some antibodies are already CyTOF-ready

• Tissue microarrays:

  • Validate CDH15 antibodies on tissue microarrays for efficient screening

  • Analyze expression across multiple samples simultaneously

• Single-cell technologies:

  • Apply validated antibodies in single-cell Western blot platforms

  • Use in droplet-based antibody screening methods

• Spatial transcriptomics:

  • Combine CDH15 protein detection with mRNA visualization

  • Correlate protein expression with transcriptional profiles in situ

What are the best practices for long-term storage and handling of CDH15 antibodies?

For optimal antibody performance and longevity:

• Storage conditions:

  • Store according to manufacturer recommendations, typically at -20°C to -80°C for long-term storage

  • Aliquot to avoid repeated freeze-thaw cycles

  • For frequent use, store at 2-8°C for up to 1 month

• Reconstitution:

  • For lyophilized antibodies, reconstitute according to manufacturer instructions

  • Use sterile buffers to prevent contamination

• Buffer considerations:

  • Many CDH15 antibodies are supplied in buffer containing glycerol (typically 50%) and preservatives like proclin-300 or sodium azide

  • Note that sodium azide inhibits HRP activity and should be avoided when using HRP-conjugated antibodies

• Stability monitoring:

  • Include consistent positive controls to monitor antibody performance over time

  • Document lot-to-lot variation if using the antibody for long-term studies

• Documentation:

  • Keep detailed records of antibody source, lot number, validation, and experimental conditions

  • This facilitates troubleshooting and reproducibility