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PCDH15 · Western blot design guide
Design a Western Blot for PCDH15
Real validated PCDH15 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PCDH15 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
PCDH15 Western Blot Experimental Design Guide
Expected bands, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~216.1 kDa | |
| Gel | 4–12% gradient | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Extensive glycosylation | |
| Regulation | IFN-γ-induced | |
| Isoform | 6 isoform(s) |
Section 1
Real Curated PCDH15 Western Blot Protocols
Literature-validated Western blot parameters for PCDH15 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human placenta , Lane 2: human U2OS , Lane 3: human Hela , Lane 4: rat brain , Lane 5: rat lung , Lane 6: mouse brain , Lane 7: mouse lung , Lane 8: mouse kidney , Lane 9: mouse spleen , Lane 10: mouse Neuro-2a . After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-PCDH15 antigen affinity purified polyclonal antibody (Catalog # A03591-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for PCDH15 at approximately 190KD. The expected band size for PCDH15 is at 216KD |
| Gel % | 4–12% gradient |
| Load | 50ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:10000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 190 kDa |
Section 2
What Is the Expected PCDH15 Western Blot Band Size?
PCDH15 has a 216 kDa predicted backbone but is expected to run higher and more diffusely due to extensive N-glycosylation across 13 sites.
| single band running above the 216 kDa predicted mass | carbohydrate added at the 13 annotated N-glycosylation sites increases the apparent mass of the mature protein |
| diffuse or smeared band rather than a sharp line | uneven glycan occupancy and branching across the many N-glycosylation sites produces mass microheterogeneity |
| several bands at different apparent masses | the 6 annotated splice isoforms (1, 2, 3, 4, 5, 7) differ in length and can be co-expressed |
| mature band slightly smaller than the full-length translated protein | cleavage of the 26-residue signal peptide removes a small N-terminal segment to generate the mature protein |
| weak or absent band in a standard whole-cell lysate | PCDH15 is a single-pass type I membrane protein and needs adequate membrane solubilization to be recovered |
💡Expected PCDH15 appearanceExpect a single, somewhat diffuse band above the 216 kDa predicted mass, broadened and shifted upward by N-glycosylation at 13 sites, with the mature form slightly smaller than the unprocessed precursor after signal-peptide cleavage.
| predicted mass (216.1 kDa, 1955 aa) | sets the baseline unmodified backbone size before any processing |
| N-glycosylation (13 sites, e.g. Asn52, Asn97, Asn201, Asn419) | adds carbohydrate mass and shifts the apparent band upward and broader relative to the 216 kDa backbone |
| signal peptide cleavage (residues 1-26) | removes the N-terminal signal sequence so the mature membrane-inserted form runs slightly smaller than the full translated precursor |
| splice isoforms (1, 2, 3, 4, 5, 7) | co-expressed isoforms of differing length can appear as multiple bands rather than one uniform band |
| single-pass type I membrane topology | incomplete membrane solubilization during lysis reduces recovered signal rather than shifting band size |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | heavy N-glycosylation across up to 13 sites adds substantial carbohydrate mass to the 216 kDa backbone | treat lysate with a deglycosylation enzyme such as PNGase F and confirm the band collapses toward the predicted mass |
| Broad smear instead of sharp band | heterogeneous glycan occupancy and branching across the many N-glycosylation sites | run a lower-percentage or gradient gel with a longer transfer time to better resolve a large, heavily glycosylated protein |
| Multiple bands | co-expression of several of the 6 annotated splice isoforms, which differ in length | check which isoforms the source tissue expresses and whether the antibody epitope falls within a region shared by all isoforms |
| No band in lysate | PCDH15 is a single-pass type I membrane protein that can remain in an insoluble membrane fraction if not properly solubilized | use a membrane-protein-compatible lysis buffer with adequate detergent and check the insoluble pellet fraction |
| Fragments below expected size | proteolytic degradation of this large, multidomain extracellular protein during sample preparation | add protease inhibitors, keep samples cold throughout lysis, and load fresh lysate promptly |
| Weak or no signal | restricted native expression (e.g., inner ear hair bundles, retina) limits PCDH15 abundance in many standard cell lysates | use an enriched native tissue lysate or an overexpression system and increase total protein loaded |
Experiment evidence
Western blot images showing PCDH15 band
A03591-1 · PCDH15
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PCDH15 Western blot
🧪For positive controls for PCDH15 in Western blot, you can use lysate from cells transiently transfected with recombinant PCDH15 (co-expressed with LHFPL5 for proper membrane trafficking), since the Human Protein Atlas provides no tissue or cell line expression data for this protein.
Positive control: PCDH15/LHFPL5-transfected cell lysate
Negative control: no HPA expression data available; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin antibody blots alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: As a single-pass type I membrane protein whose surface localization depends on LHFPL5 co-expression, with no HPA tissue data to guide a positive/negative pair, reliable controls require an engineered overexpression system paired with siRNA/KO validation rather than native tissue lysates.
Comprehensive Human Protein Atlas IHC scoring per tissue. Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced PCDH15 Western Blot Tips
Deeper troubleshooting and optimisation questions for PCDH15, answered from its protein features.
Why does PCDH15 run higher than its predicted 216 kDa mass?
PCDH15 carries 13 predicted N-glycosylation sites, and glycosylation typically increases apparent molecular weight on SDS-PAGE. Expect the observed band to migrate above the 216 kDa calculated mass, sometimes as a broad or smeared band from heterogeneous glycan occupancy across the ectodomain's many cadherin repeats.
Will PCDH15 isoforms produce multiple Western blot bands?
UniProt lists six annotated isoforms (1, 2, 3, 4, 5, 7) generated by alternative splicing, so antibodies targeting shared exons can detect several bands of differing size depending on tissue and splice variant expressed. Confirm the antibody epitope's exon location relative to spliced regions before interpreting multiple bands as isoforms versus degradation.
Does glycosylation affect PCDH15 band sharpness or mobility?
With 13 glycosylation sites, PCDH15 may show a diffuse or fuzzy band rather than a sharp line, since glycan occupancy and branching vary across molecules. Treating lysates with PNGase F before loading removes N-linked glycans, sharpens the band, and confirms that the shift and smearing are glycosylation-dependent rather than degradation.
How should blocking be optimized for a glycoprotein like PCDH15?
PCDH15 is heavily glycosylated, and non-fat milk contains glycoproteins that can cross-react with glycan-directed reagents or contribute nonspecific background on glycoprotein targets. Use BSA-based blocking buffer instead of milk to minimize this interference, and extend blocking time given the protein's large ectodomain and multiple glycosylation sites requiring thorough antibody access.
What transfer method to use for PCDH15 Western blot?
At approximately 216 kDa plus glycosylation, PCDH15 is a large membrane protein that transfers poorly with standard rapid systems. Use wet (tank) transfer with reduced methanol (10%) transfer buffer, run a low-percentage (5-6%) or gradient SDS-PAGE gel, and transfer overnight at low voltage or several hours at higher voltage for efficient high-molecular-weight protein transfer.
What loading controls suit quantifying membrane-bound PCDH15?
PCDH15 is a single-pass type I membrane protein requiring LHFPL5 for efficient plasma membrane localization, so extract with membrane-compatible detergents to solubilize the full receptor. Normalize band intensity to a membrane-resident loading control or total protein stain rather than a cytosolic housekeeping protein, since membrane fraction recovery efficiency can vary between samples.
What explains lower-molecular-weight bands on a PCDH15 blot?
PCDH15 forms an antiparallel heterodimer with CDH23 and interacts with TMIE, LHFPL5, and MYO7A, stabilized partly by a single disulfide bond. Under non-reducing conditions, complex-associated or disulfide-linked species may appear; reducing sample buffer should resolve monomeric PCDH15. Smaller unexpected bands more often reflect proteolytic degradation or incomplete extraction than genuine isoforms.
Boster reagents
Best PCDH15 Western Blot Antibodies
BosterBio's PCDH15 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-PCDH15 Antibody Picoband®
Cat # A03591-1
Our recommended anti-PCDH15 western blot antibody is a best-in-class, extensively cited reagent, thoroughly validated by SDS-PAGE western blotting and orthogonally cross-validated against negative-control tissues and complementary detection methods for confident, reproducible PCDH15 protein detection.
Which to pick: Only one Boster PCDH15 antibody is catalogued, A03591-1, which includes a real SDS-PAGE western blot validation image, making it the clear, straightforward choice for your PCDH15 western blot experiments.
Source: BosterBio PCDH15 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q96QU1.
- Human Protein Atlas. PCDH15 tissue expression.