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- Table of Contents
GPX3 / Glutathione peroxidase 3 · Western blot design guide
Design a Western Blot for GPX3
Real validated GPX3 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-GPX3 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled September 2026 · For research use; verify linked source records and product datasheet before use
GPX3 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 | ~25.6 kDa | |
| Gel | 12–15% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Cleaved | |
| Caveat | Secreted protein | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated GPX3 Western Blot Protocols
The A02637 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | lysates from human kidney, plasma, mouse kidney, plasma and rat kidney tissue lysate (from left to right), (catalog A02637) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A02637; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | goat anti-rabbit IgG, 1:5000 (catalog A02637) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected GPX3 Western Blot Band Size?
GPX3 has a predicted 25.6 kDa precursor; signal-peptide cleavage could lower mature mass, but no empirical migration size or distinct band pattern is established.
| Band near 25.6 kDa | compatible with the predicted full-length GPX3 precursor; confirm identity with controls |
| Band below 25.6 kDa | could reflect removal of the signal peptide; mature migration is not established |
| Little or no band in whole-cell lysate | compatible with secretion of GPX3 |
| Band in plasma | compatible with secreted GPX3; confirm identity with controls |
💡Expected GPX3 appearanceUniProt predicts a 25.6 kDa full-length precursor; signal-peptide cleavage may lower mature GPX3 migration, but no empirical band size is supplied, so confirm band identity with ordinary controls.
| Predicted full-length mass | 25.6 kDa is the precursor reference |
| Signal peptide at residues 1–20 | its removal lowers mature protein mass |
| Full-length precursor | retains the signal peptide and is larger than the cleaved product |
| Mature protein after signal-peptide cleavage | may migrate below the precursor; its apparent size is unmeasured |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | GPX3 is secreted | check plasma or conditioned medium and verify sample loading |
| Band lower than expected | signal-peptide removal is possible | compare with a positive control and verify band identity |
| Band higher than expected | the supplied features do not establish a higher-migrating form | check specificity with a validated positive control |
| Multiple bands | precursor and mature protein are possible, but distinct bands are unverified | compare cellular and secreted samples and check antibody specificity |
| Weak or no signal | secreted GPX3 may be scarce in the sampled lysate | test plasma or conditioned medium with a positive control |
Experiment evidence
Western blot images showing GPX3 band
A02637 · GPX3
A02637 · GPX3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for GPX3 Western blot
🧪For positive controls for GPX3 in Western blot, you can use a verified GPX3-positive conditioned-medium sample; no HPA positive tissue or cell is supplied.
Positive control: No high/medium HPA tissue identified
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: GPX3 is secreted, so conditioned medium may be more suitable than whole-cell lysate.
HPA tissue expression evidence for GPX3
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.
Higher expression tissues · candidate positive controls from IHC
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No high/medium HPA tissues identified in the supplied evidence. | ||||
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No lower-expression tissue rows available in the supplied evidence. | ||||
Section 3
Advanced GPX3 Western Blot Tips
Deeper troubleshooting and optimisation questions for GPX3, answered from its protein features.
How should GPX3 band migration be interpreted?
Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.
Are multiple GPX3 isoforms expected from these features?
Isoforms · Only one isoform is listed, with no alternative sequence. These features do not provide an isoform-based explanation for multiple bands.
Do the supplied features identify a GPX3 glycosylation or modification site?
PTM · No glycosylation sites or modified residues are listed. The sequence contains selenocysteine, and positions 1–20 are annotated as a signal peptide. These facts alone do not demonstrate a visible band shift.
Does this guide establish induction of GPX3?
Induction · No general induction response is established by this guide. A pathway or gene-set association is not evidence of induction in a particular specimen. Verify the relevant treatment and control in a target-specific experiment.
What transfer method to use for GPX3 Western blot?
Transfer · The features specify a predicted mass of 25.6 kDa but no transfer method. Check transfer and membrane retention around that size using a molecular weight marker and an appropriate transfer control.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02637 datasheet where specified. Otherwise compare 5% milk or 5% BSA in TBST; for a phospho-specific assay start with BSA. Optimize background and specific signal with matched controls.
How should GPX3 be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Why might GPX3 migrate differently from its predicted 25.6 kDa?
Interpretation · The 25.6 kDa prediction is for the 226-residue sequence, which includes a signal peptide at UniProt positions 1–20. Signal peptide processing could change the mass of secreted GPX3, but these features do not establish its apparent band position.
GPX3 is annotated as secreted. Include the extracellular fraction, such as conditioned medium, when choosing samples; a cell lysate alone may miss secreted protein.
GPX3 is annotated as a homotetramer. Consider oligomerization when investigating a higher band, but the annotation does not show that a tetramer survives Western blot preparation or specify where it would migrate.
Compare their positions with the 25.6 kDa full-sequence prediction while accounting for the signal peptide at UniProt positions 1–20. The secreted location and homotetramer annotation offer possibilities to investigate, but no observed band or feature here identifies a particular unexpected band.
GPX3 is annotated as secreted. Include the extracellular fraction, such as conditioned medium, when choosing samples; a cell lysate alone may miss secreted protein.
GPX3 is annotated as a homotetramer. Consider oligomerization when investigating a higher band, but the annotation does not show that a tetramer survives Western blot preparation or specify where it would migrate.
Compare their positions with the 25.6 kDa full-sequence prediction while accounting for the signal peptide at UniProt positions 1–20. The secreted location and homotetramer annotation offer possibilities to investigate, but no observed band or feature here identifies a particular unexpected band.
Boster reagents
GPX3 Western Blot Antibodies
Catalog antibodies with Western blot application and product-specific WB images. Evaluate suitability with the reported sample, controls and experimental conditions.
Real WB data
Anti-GPX3 Antibody (Center)
Cat # A02637
The catalog reports A02637, Anti-GPX3 Antibody (Center), for Western blot with stated human and mouse reactivity. Its WB image caption reports human and mouse kidney and plasma lysates, plus rat kidney tissue lysate, at 35 µg per lane and 1:1000 primary dilution.
Which to pick: A02637 is the only listed option and has a WB image. Choose it for human or mouse samples based on stated reactivity and the reported kidney and plasma examples. Rat kidney appears in the caption, but rat is not listed under reactivity.
Source: BosterBio GPX3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P22352.
- Human Protein Atlas. GPX3 tissue expression.
- PMC4990756 — target-verified WB comparison
- PMC8948607 — target-verified WB comparison
- PMC12291489 — target-verified WB comparison