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PHEX · Western blot design guide
Design a Western Blot for PHEX
Real validated PHEX Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PHEX WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
PHEX 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 | ~86.5 kDa | |
| Observed band | 100-110 kDa | |
| Gel | 8–10% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated | |
| Caveat | Heavy glycosylation | |
| Regulation | Complement | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated PHEX Western Blot Protocols
Literature-validated Western blot parameters for PHEX — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human PC-3 , Lane 2: human U251 , Lane 3: human A431 , Lane 4: human A549 , After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA 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-PHEX antigen affinity purified polyclonal antibody (A02078) 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 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for PHEX at approximately 100-110 kDa. The expected band size for PHEX is at 86 kDa |
| Gel % | 8–10% |
| Load | 30 ug |
| 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, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 100–110 kDa |
Section 2
What Is the Expected PHEX Western Blot Band Size?
PHEX has an 86.5 kDa predicted core but runs at 100-110 kDa on Western blot because its 8 N-linked glycosylation sites add substantial carbohydrate mass.
| single band around 100-110 kDa | mature, fully glycosylated PHEX running well above its 86.5 kDa predicted core mass |
| broad or fuzzy band spanning roughly 90-110 kDa | heterogeneous occupancy across the 8 N-linked glycosylation sites gives a mixed population of glycoforms |
| band closer to 86.5 kDa | corresponds to the unmodified or deglycosylated PHEX backbone rather than the mature membrane-bound form |
| little or no signal from a cytosolic-only prep | PHEX is a single-pass type II membrane protein and needs membrane-compatible lysis to be released and detected |
| band shifts slightly with reducing vs non-reducing conditions | the 5 intrachain disulfide bonds stabilize PHEX's extracellular fold, so incomplete reduction can alter apparent migration |
💡Expected PHEX appearancePHEX typically appears as a band at 100-110 kDa, above its 86.5 kDa predicted mass, because the mature type II membrane glycoprotein carries N-glycans at 8 sites.
| predicted core mass (86.5 kDa) | sets the unmodified backbone size before any glycosylation is added |
| N-glycosylation at 8 asparagine sites (e.g., Asn71, Asn238, Asn377, Asn736) | adds carbohydrate mass, pushing the observed band up to roughly 100-110 kDa and potentially broadening it |
| single-pass type II membrane topology | hydrophobic transmembrane segment can cause anomalous SDS-PAGE migration and requires membrane-protein extraction for full solubilization |
| intrachain disulfide bonds (5 pairs stabilizing the extracellular domain) | maintain a compact fold that, if not fully reduced, can shift apparent band position on the gel |
| non-glycosylated or bacterially-expressed recombinant PHEX | lacks the native N-glycans and runs closer to the 86.5 kDa predicted mass, lower than tissue-derived PHEX |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | PHEX is membrane-anchored and is poorly recovered by cytosolic-only lysis | use a membrane-protein extraction buffer with adequate detergent to solubilize the transmembrane protein |
| Band higher than expected | incomplete denaturation of the glycosylated membrane protein or full glycan loading | ensure thorough reduction and denaturation with DTT/β-mercaptoethanol and SDS before loading |
| Band lower than expected | reduced or absent N-glycosylation, as in deglycosylated samples or non-native recombinant expression | compare against a native glycosylated tissue lysate control and note the 86.5 kDa unmodified backbone as reference |
| Broad smear instead of sharp band | heterogeneous glycan occupancy across the 8 N-linked sites | treat lysate with PNGase F to collapse glycoforms toward the 86.5 kDa core band |
| Weak or no signal | PHEX expression is restricted to tissues such as bone, kidney, and teeth, so many cell lines express little of it | use a known PHEX-expressing lysate as a positive control and increase total protein loaded |
| Multiple bands | partial N-glycan occupancy or proteolytic degradation of the membrane protein during extraction | add protease inhibitors during lysis and run a PNGase F-treated sample alongside to distinguish glycoform bands from degradation fragments |
Experiment evidence
Western blot images showing PHEX band
A02078 · PHEX
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PHEX Western blot
🧪For positive controls for PHEX in Western blot, you can use lysate from a PHEX-transfected or overexpressing cell line, since no confirmed high-expressing tissue is available from the current evidence.
Positive control: PHEX-overexpressing cell lysate
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Also probe for GAPDH and β-actin alongside a total-protein stain such as stain-free imaging, Ponceau S, or REVERT to confirm equal loading.
⚠️Feasibility: As a single-pass type II membrane protein with no HPA expression data to guide a reliable endogenous positive/negative tissue pair, use membrane-enriched lysis and validate specificity with an overexpression construct plus a knockdown/knockout line.
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 PHEX Western Blot Tips
Deeper troubleshooting and optimisation questions for PHEX, answered from its protein features.
Why does PHEX run at 100-110 kDa instead of 86.5 kDa?
PHEX carries 8 N-glycosylation sites; added sugar chains add roughly 15-25 kDa to the 86.5 kDa core protein, so denaturing SDS-PAGE shows the 100-110 kDa mature glycoprotein rather than the unmodified core mass. This shift is expected and confirms proper glycosylation, not degradation or nonspecific binding.
Should multiple bands be expected for PHEX?
UniProt lists only one PHEX isoform, so a single specific band near 100-110 kDa is expected. Extra bands likely reflect antibody cross-reactivity, incomplete deglycosylation, or degradation fragments below 86.5 kDa rather than genuine isoforms, since no alternative splice variants are annotated for this gene.
How does glycosylation affect PHEX antibody detection?
With 8 N-glycosylation sites, the mature PHEX glycoform (100-110 kDa) may show reduced signal if the antibody epitope overlaps a glycosylated region. If signal is weak or diffuse, treat lysates with PNGase F to collapse the band toward the 86.5 kDa core and verify antibody specificity.
What blocking approach avoids interfering with PHEX detection?
Standard BSA or non-fat milk blocking works for conventional antibody-based PHEX detection. Prefer BSA over milk if using lectin- or biotin-based glycan detection alongside antibody probing, since milk's endogenous glycoproteins and biotin can add background given PHEX's heavily glycosylated, membrane-anchored nature.
What transfer method to use for PHEX Western blot?
As an 86.5 kDa single-pass type II membrane protein that migrates at 100-110 kDa once glycosylated, PHEX transfers best with wet tank transfer at low voltage over an extended time. This handles large, disulfide-stabilized membrane glycoproteins more reliably than rapid semi-dry systems.
Should samples be reduced for accurate PHEX quantitation?
Yes. PHEX has 5 disulfide bonds and forms a zinc-dependent complex with MEPE via the ASARM motif. Use DTT or beta-mercaptoethanol to fully reduce samples before SDS-PAGE so PHEX migrates consistently at 100-110 kDa, preventing disulfide-linked aggregates from skewing band quantitation.
What could explain an unexpected higher-molecular-weight PHEX band?
PHEX interacts with MEPE in a zinc-dependent manner via the ASARM motif; incomplete reduction or non-denaturing conditions can preserve this complex or disulfide-linked oligomers, producing bands above 100-110 kDa. Confirm by reducing and denaturing fully, and include zinc chelators if complex formation is suspected.
Do PHEX disease variants alter expected band size?
PHEX is annotated with disease variants linked to X-linked hypophosphatemia, some of which are truncating mutations. In patient-derived samples, a band migrating well below 100 kDa may indicate a pathogenic truncated protein product rather than a loading, transfer, or antibody artifact.
Boster reagents
Best PHEX Western Blot Antibodies
BosterBio's PHEX 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-PHEX Antibody Picoband®
Cat # A02078
These top-performing anti-PHEX antibodies are extensively cited and rigorously validated, with specificity confirmed through negative-tissue controls and complementary orthogonal methods, ensuring reliable, reproducible Western blot detection of PHEX.
Which to pick: Only one anti-PHEX antibody is catalogued here, A02078, and it comes with an actual Western blot validation image (8% SDS-PAGE), making it the clear default pick—no alternative SKUs are currently listed to compare against.
Source: BosterBio PHEX gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P78562.
- Human Protein Atlas. PHEX tissue expression.