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- Table of Contents
LOX · Western blot design guide
Design a Western Blot for LOX
Real validated LOX Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-LOX WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
LOX 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 | ~46.9 kDa | |
| Observed band | ~47 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 | Propeptide cleavage | |
| Regulation | Hypoxia up | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated LOX Western Blot Protocols
Literature-validated Western blot parameters for LOX — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | rat liver , Lane 2: rat lung , Lane 3: mouse liver . 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-LOX antigen affinity purified polyclonal antibody (Catalog # PB9718) 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 Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for LOX at approximately 47 kDa. The expected band size for LOX is at 47 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 | 0.5 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 | 47 kDa |
Section 2
What Is the Expected LOX Western Blot Band Size?
LOX has a 46.9 kDa predicted mass and runs at the empirically observed ~47 kDa as the secreted proenzyme, with propeptide N-glycosylation accounting for the slight upward shift.
| single band near 47 kDa | matches the secreted full-length LOX proenzyme after signal peptide cleavage, close to the 46.9 kDa predicted mass |
| band running slightly above the 46.9 kDa predicted mass | N-linked glycosylation at three sites (Asn81, Asn97, Asn144) within the retained propeptide adds modest mass to the secreted proenzyme |
| little or no band in whole-cell lysate | LOX is a secreted, extracellular protein, so most of it leaves the cell rather than staying in the cytoplasmic fraction |
| smaller ~30 kDa and ~18 kDa bands appearing alongside or instead of the ~47 kDa band | extracellular proteolytic cleavage between the propeptide (22-168) and the mature chain (169-417) separates a catalytic fragment from a propeptide fragment |
| slightly diffuse or fuzzy edge on the ~47 kDa band | partial or heterogeneous occupancy of the three N-glycosylation sites in the propeptide produces minor size heterogeneity |
💡Expected LOX appearanceLOX typically appears as a single band at approximately 47 kDa, consistent with the 46.9 kDa predicted mass of the secreted proenzyme carrying an intact, lightly glycosylated propeptide.
| Predicted mass (UniProt, 417 aa) | sets the 46.9 kDa reference point for the intact secreted proenzyme |
| Signal peptide cleavage (residues 1-21) | removed upon secretion, so the extracellular/lysate-detectable form runs slightly lower than the unprocessed intracellular precursor |
| Propeptide retention vs cleavage (residues 22-168) | an intact propeptide keeps the secreted band near the full ~47 kDa size, while its extracellular removal produces a smaller mature catalytic fragment |
| N-linked glycosylation at Asn81, Asn97, Asn144 | adds modest mass and can broaden the band slightly above the unmodified predicted size |
| Secreted/extracellular localization | protein is depleted from standard whole-cell lysate and better recovered from conditioned media, ECM-enriched, or tissue fractions |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | LOX is secreted into the extracellular space/matrix rather than retained inside the cell | probe conditioned media, ECM-enriched or decellularized matrix fractions, or whole tissue lysates instead of standard cytoplasmic lysate |
| Band higher than expected | N-glycosylation at the three propeptide sites adds mass to the secreted proenzyme | treat the sample with PNGase F and confirm the band shifts toward the 46.9 kDa predicted mass |
| Fragments below expected size | extracellular proteolytic maturation cleaves the propeptide (22-168) from the mature chain (169-417), releasing smaller species | add protease inhibitors during extraction and interpret smaller fragments as physiological maturation products rather than degradation |
| Broad smear instead of sharp band | heterogeneous occupancy of the three N-glycosylation sites in the propeptide | run deglycosylated (PNGase F-treated) and untreated samples side by side to sharpen and confirm the band |
| Weak or no signal | standard lysis buffers are not designed to capture secreted, matrix-associated LOX | concentrate conditioned media or use matrix-extraction methods and load higher total protein |
| Multiple bands | coexistence of the full-length ~47 kDa proenzyme with cleaved mature and propeptide fragments in tissue samples | compare across tissues and, if available, use antibodies targeting the propeptide versus the mature domain to assign each band |
Experiment evidence
Western blot images showing LOX band
PB9718 · LOX
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for LOX Western blot
🧪For positive controls for LOX in Western blot, you can use a recombinant LOX-overexpression lysate or conditioned medium, since no tissue- or cell-line-specific expression data is available from HPA for this protein.
Positive control: Overexpression lysate/conditioned medium
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin antibody controls alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) for normalization.
⚠️Feasibility: As a secreted protein with no HPA expression data available, whole-cell lysate signal may be weak, so favor conditioned medium as the sample source and confirm band identity with siRNA knockdown or a KO 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 LOX Western Blot Tips
Deeper troubleshooting and optimisation questions for LOX, answered from its protein features.
Why does LOX run near its predicted 46.9 kDa mass?
The observed ~47 kDa band matches the secreted proenzyme: the 21-residue signal peptide is cleaved but the 22-168 propeptide is retained prior to extracellular processing. This full-length precursor mass aligns closely with the 46.9 kDa prediction, so the antibody is detecting the unprocessed secreted form rather than a proteolytically matured fragment.
How does TPQ formation affect LOX gel mobility?
TPQ (topaquinone) is a catalytic cofactor generated post-translationally from an internal tyrosine, one of LOX's two modified residues. It adds no significant mass and is not removed by SDS-PAGE denaturation, so it does not shift the observed band; the ~47 kDa signal reflects the polypeptide backbone, not cofactor status.
Does copper binding change LOX's apparent molecular weight?
Copper is a tightly but non-covalently bound catalytic metal cofactor. Under denaturing SDS-PAGE conditions it dissociates from the protein and contributes no detectable mass, so occupancy status does not shift the band position. If preserving copper-dependent activity for downstream assays, avoid harsh chelation during lysis.
What blocking buffer best suits LOX's three glycosylation sites?
LOX carries three N-glycosylation sites, so BSA-based blocking is preferable to milk, which contains glycoproteins and lectins that can cross-react with glycosylated epitopes and raise background. This is especially important if the primary antibody's epitope lies near a glycosylated region of the secreted protein.
What transfer method to use for LOX Western blot?
Standard wet or semi-dry transfer works well at ~47 kDa. Because LOX is a secreted glycoprotein stabilized by five disulfide bonds, run samples under fully reducing and denaturing conditions before transfer, and consider extended transfer time to compensate for the compact, disulfide-constrained tertiary structure.
Which loading control fits LOX's secreted localization?
Since LOX is secreted extracellularly rather than retained intracellularly, standard housekeeping controls like GAPDH or actin are inappropriate for conditioned-media samples. Use a total protein stain such as Ponceau S for normalization, or a validated secreted-protein control, when quantifying LOX in extracellular fractions.
Why do extra bands appear below the 47 kDa band?
LOX is secreted as a proenzyme with a propeptide spanning residues 22-168 that is proteolytically removed extracellularly to yield the active mature enzyme. Antibodies raised against the catalytic domain versus the propeptide region will detect different processed fragments, producing additional lower-molecular-weight bands beyond the full-length precursor.
Boster reagents
Best LOX Western Blot Antibodies
BosterBio's LOX 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-LOX Antibody Picoband®
Cat # PB9718
Our top-performing anti-LOX antibodies are extensively cited and rigorously validated, with specificity confirmed via orthogonal methods including negative-tissue controls and complementary detection techniques, ensuring reliable, reproducible Western blot results for LOX detection across applications.
Which to pick: Only one Boster anti-LOX antibody is currently catalogued, PB9718, which includes an actual Western blot validation image on a 5-20% SDS-PAGE gel, making it the clear default choice for LOX WB experiments.
Source: BosterBio LOX gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P28300.
- Human Protein Atlas. LOX tissue expression.