This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
CPO · Western blot design guide
Design a Western Blot for CPO
Real validated CPO Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CPO WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
CPO 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 | ~42.5 kDa | |
| Observed band | ~57 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | N-glycosylation adds mass | |
| Regulation | LPS-induced | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated CPO Western Blot Protocols
Literature-validated Western blot parameters for CPO — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human Caco-2 , Lane 2: human COLO-320 , Lane 3: human SW620 , Lane 4: human AGS . 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-CPO antigen affinity purified polyclonal antibody (Catalog # A03649-1) at 0.25 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 Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for CPO at approximately 57 kDa. The expected band size for CPO is at 43 kDa |
| Gel % | 10–12% |
| 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 | 0.25 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 | 57 kDa |
Section 2
What Is the Expected CPO Western Blot Band Size?
CPO has a 42.5 kDa predicted backbone but runs at approximately 57 kDa on Western blot, mainly due to N-glycosylation at four asparagine sites.
| single band at approximately 57 kDa | the mature, glycosylated, GPI-anchored form of CPO, running well above its 42.5 kDa predicted precursor mass |
| broad, diffuse band spanning roughly 55-65 kDa instead of a sharp line | heterogeneous glycan occupancy across the four N-glycosylation sites (Asn132, Asn174, Asn187, Asn251) creates a mixture of glycoforms |
| faint fainter band running below the main ~57 kDa band | a less fully glycosylated intermediate, or a form still retaining part of the cleaved signal peptide/propeptide before complete maturation |
| weak or absent band in soluble/cytosolic lysate fractions | CPO is GPI-anchored at the apical cell membrane, so it concentrates in membrane preparations rather than cytosolic lysate |
| single band with no companion band at roughly twice its size | no disulfide bonds are annotated for CPO, so it is not expected to form a covalent homodimer |
| single band with no extra bands at other molecular weights | UniProt lists only one isoform for CPO, so multiple bands from alternative splicing are not expected |
💡Expected CPO appearanceExpect a single band near 57 kDa, the empirically observed size, running above the 42.5 kDa predicted mass because N-glycosylation at four asparagine sites outweighs the small mass loss from signal peptide and propeptide cleavage.
| Predicted backbone mass (UniProt) | 42.5 kDa is the unmodified sequence mass; the native band observed at ~57 kDa runs well above this once modifications are added |
| N-glycosylation at Asn132, Asn174, Asn187, Asn251 | combined glycan mass at these four sites is the main driver pushing the band from 42.5 kDa up toward the ~57 kDa observed size, and can broaden it into a smear |
| Signal peptide cleavage (residues 1-20) | removal during maturation trims a small amount of mass, but this loss is masked by the larger glycosylation gain |
| Propeptide cleavage (residues 353-374) | processing of the C-terminal propeptide yields the mature form and modestly reduces mass relative to the unprocessed precursor |
| GPI-anchor and apical membrane localization | band detection depends on sample prep enriching the membrane fraction, since the protein partitions there rather than into soluble lysate |
| Non-glycosylated recombinant standard | a bacterially expressed standard lacking N-glycans would run closer to the 42.5 kDa predicted mass rather than the ~57 kDa native band |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CPO is GPI-anchored at the apical cell membrane, so standard cytosolic-focused lysis can under-represent it | use a lysis buffer and detergent that effectively solubilize membrane and GPI-anchored proteins, and include a tissue/cell line known to express CPO as a positive control |
| Band higher than expected | N-glycosylation at four asparagine residues adds mass above the 42.5 kDa predicted backbone, producing the ~57 kDa native band | treat a parallel sample with PNGase F and check for a shift toward the 42.5 kDa predicted mass to confirm the identity of the upshifted band |
| Broad smear instead of sharp band | heterogeneous glycan occupancy across the four N-glycosylation sites produces a mixture of glycoforms | run a lower percentage gel with longer separation, or deglycosylate the sample before electrophoresis to sharpen the band |
| Fragments below expected size | cleavage of the signal peptide and C-terminal propeptide during normal maturation removes some mass, and proteolysis during sample handling can generate further smaller fragments | add protease inhibitors during lysis and compare band size against the expected mature and precursor sizes to distinguish normal processing from degradation |
| Weak or no signal | low CPO expression or insufficient enrichment of the membrane fraction where the GPI-anchored protein resides | load more total protein, enrich for membrane proteins, and confirm detection using a known positive-control tissue or cell line |
| Multiple bands | partially versus fully glycosylated glycoforms, or precursor versus mature cleaved species, can resolve as distinct bands rather than a single one | treat with PNGase F to collapse glycoform bands to a single deglycosylated species and compare sizes to distinguish precursor from mature protein |
Experiment evidence
Western blot images showing CPO band
A03649-1 · CPO
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CPO Western blot
🧪For positive controls for CPO in Western blot, you can use lysate from a CPO-transfected/overexpressing cell line, since the supplied HPA data contains no tissue or cell expression calls to identify an endogenous positive source.
Positive control: CPO-transfected overexpression cells
Negative control: no HPA expression data; use siRNA knockdown or KO line
Loading controls: Probe the same blot for GAPDH and β-actin, and include a total-protein stain (stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: As a GPI-anchored apical-membrane protein with no HPA expression profile available, a native tissue-based positive/negative pair cannot be identified, so rely on an overexpression system with membrane-enriched lysis (e.g., detergent or PI-PLC extraction) to confirm band specificity.
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 CPO Western Blot Tips
Deeper troubleshooting and optimisation questions for CPO, answered from its protein features.
Why does CPO migrate above its predicted 42.5 kDa mass?
CPO carries 4 predicted N-glycosylation sites and a GPI-anchor, both of which add mass and alter SDS-PAGE mobility. The added carbohydrate and lipid-linked glycan structures commonly cause carboxypeptidases to run heavier than their unmodified sequence mass, consistent with the ~57 kDa band observed against the 42.5 kDa prediction.
Should multiple isoform bands be expected for CPO?
UniProt lists only one CPO isoform, so a single principal band is expected. Additional bands more likely reflect precursor-to-mature processing (signal peptide and propeptide removal) or partial glycosylation rather than alternative splicing products.
How does propeptide removal affect the CPO band pattern?
CPO is synthesized with a cleavable signal peptide (residues 1-20) and a propeptide (residues 353-374) removed during activation. Blots may show a slightly larger zymogen form alongside the smaller mature enzyme, so both bands can be biologically valid rather than indicating degradation.
How should blocking be optimized for CPO detection?
Because CPO is a glycoprotein with 4 glycosylation sites, casein-based milk blockers can introduce lectin-like background from milk glycoproteins. Use a BSA-based blocking buffer instead to minimize nonspecific carbohydrate interactions and improve signal specificity for this glycosylated target.
What transfer method to use for CPO Western blot?
CPO is a GPI-anchored apical membrane protein, so include SDS in the transfer buffer and use wet transfer to fully elute the lipid-anchored protein from the gel. A standard methanol-containing Tris-glycine buffer with PVDF membrane is suitable, as no disulfide-linked oligomeric complexes are reported.
What extraction step improves CPO quantitation accuracy?
Since CPO is GPI-anchored to the apical cell membrane, standard RIPA lysis may leave residual membrane-associated protein unextracted. Include a detergent step (e.g., Triton X-100) or PI-PLC treatment to fully solubilize GPI-anchored CPO before loading, ensuring quantitation reflects total protein rather than a partially extracted fraction.
What explains extra bands above or below the main CPO band?
A higher band may represent the uncleaved zymogen retaining its propeptide, while a lower band could reflect autoproteolysis, since CPO is an active zinc metalloprotease. Variable glycan occupancy across the 4 glycosylation sites can also produce a diffuse or doublet appearance rather than a single sharp band.
Boster reagents
Best CPO Western Blot Antibodies
BosterBio's CPO 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-CPO Antibody Picoband®
Cat # A03649-1
Our recommended anti-CPO antibody is a top-performing, extensively cited reagent, thoroughly validated across expression systems and orthogonally cross-checked against negative-control tissue and complementary detection methods to confirm specificity and reliable Western blot performance.
Which to pick: Only one anti-CPO antibody is catalogued here, A03649-1, and it includes an actual Western blot validation image, so it is both the recommended and the only option for confirming CPO detection in your samples.
Source: BosterBio CPO gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q8IVL8.
- Human Protein Atlas. CPO tissue expression.