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- Table of Contents
DIO3 / Thyroxine 5-deiodinase · Western blot design guide
Design a Western Blot for DIO3
Real validated DIO3 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-DIO3 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
DIO3 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 | ~33.9 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 | — | |
| Caveat | Oligomerization controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated DIO3 Western Blot Protocols
The A02903 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | various cells (catalog A02903) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A02903; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| 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 DIO3 Western Blot Band Size?
DIO3 has a predicted monomer mass of 33.9 kDa; homodimerization could affect migration, but no empirical band or demonstrated shift is supplied.
| Band near 33.9 kDa | Consistent with the predicted DIO3 monomer; confirm its identity with controls |
| Band near twice 33.9 kDa | Could reflect a DIO3 homodimer if the complex survives electrophoresis |
| Bands near 33.9 kDa and twice that size | Could reflect monomeric and retained homodimeric DIO3 |
| Little signal in a soluble lysate fraction | DIO3 is a cell and endosome membrane protein |
💡Expected DIO3 appearanceThe predicted DIO3 monomer is 33.9 kDa; no empirical band size is supplied, and band identity requires appropriate antibody and sample controls.
| UniProt predicted molecular mass | Places the DIO3 monomer near 33.9 kDa |
| Monomeric DIO3 | Provides the predicted single-protein band position |
| DIO3 homodimer formation | Could yield a band near twice the monomer mass if the complex persists during electrophoresis |
| Homodimer stability during sample preparation | Determines whether a higher band from the documented homodimer remains detectable |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated DIO3 may be underrepresented in the sampled fraction | Check membrane enrichment and extraction, then use a positive control |
| Band higher than expected | A documented DIO3 homodimer may persist during electrophoresis | Compare sample preparation conditions and verify identity with antibody controls |
| Band lower than expected | The supplied features do not establish a smaller DIO3 product | Check antibody specificity and sample integrity |
| Multiple bands | Monomeric and homodimeric DIO3 could contribute if the dimer persists | Compare sample preparation conditions and verify each band with controls |
| Weak or no signal | DIO3 may be poorly recovered from cell or endosome membranes | Check membrane extraction, loading, and a positive control |
| Fragments below expected size | Sample degradation is possible; no cleavage product is specified | Check sample handling and confirm band identity with antibody controls |
Experiment evidence
Western blot images showing DIO3 band
A02903 · DIO3
A02903 · DIO3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for DIO3 Western blot
🧪For positive controls for DIO3 in Western blot, you can use no HPA-supported positive sample because no tissue or cell data were 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: DIO3 is membrane-associated, and the supplied HPA record identifies no validated positive or negative tissue.
HPA tissue expression evidence for DIO3
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 DIO3 Western Blot Tips
Deeper troubleshooting and optimisation questions for DIO3, answered from its protein features.
How should DIO3 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.
Could DIO3 isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It does not support assigning additional bands to DIO3 isoforms.
Do annotated modifications prove a band shift?
PTM · The linked UniProt record describes protein features. A modification annotation alone does not demonstrate a visible shift; retain any condition or experimental qualifier attached to it.
Does this guide establish induction of DIO3?
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 DIO3 Western blot?
Transfer · DIO3 is a predicted 33.9-kDa, single-pass membrane protein. Check that the transfer captures proteins in this size range. The supplied features do not identify a preferred transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02903 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 DIO3 bands be quantified?
Quantitation · Quantify a consistently identified band across comparable membrane-containing samples. Because DIO3 can be monomeric or homodimeric, document which band is measured; do not combine bands without establishing their identities.
Should DIO3 migrate at its predicted 33.9 kDa?
Interpretation · 33.9 kDa is the predicted mass; no observed band size is supplied. Use it as a reference, but determine apparent mass from the blot. The listed features alone cannot explain a difference.
DIO3 is reported as both a monomer and a homodimer, with possible minor heterodimerization with DIO1 or DIO2. Consider oligomerization when evaluating higher-mass bands, but these features do not establish that any particular band is an oligomer.
DIO3 is a single-pass type II membrane protein reported at the cell membrane and endosome membrane. Include membrane-containing material when assessing detection or comparing samples.
The canonical DIO3 sequence contains selenocysteine, but its presence alone does not demonstrate a visible band shift or explain an unexpected apparent mass. Compare any band with the predicted 33.9 kDa and verify its identity before assigning a cause.
DIO3 is reported as both a monomer and a homodimer, with possible minor heterodimerization with DIO1 or DIO2. Consider oligomerization when evaluating higher-mass bands, but these features do not establish that any particular band is an oligomer.
DIO3 is a single-pass type II membrane protein reported at the cell membrane and endosome membrane. Include membrane-containing material when assessing detection or comparing samples.
The canonical DIO3 sequence contains selenocysteine, but its presence alone does not demonstrate a visible band shift or explain an unexpected apparent mass. Compare any band with the predicted 33.9 kDa and verify its identity before assigning a cause.
Boster reagents
DIO3 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-Thyroxine 5-deiodinase DIO3 Antibody
Cat # A02903
The catalog reports one anti-DIO3 antibody for Western blot, A02903, with stated human, mouse, and rat reactivity. A WB image is listed, but the supplied description does not identify the cells or experimental conditions.
Which to pick: A02903 is the only listed option. Its stated reactivity covers human, mouse, and rat, and it has a WB image described as analysis of various cells. Check the full image details before matching it to your sample.
Source: BosterBio DIO3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P55073.
- Human Protein Atlas. DIO3 tissue expression.
- PMC9773011 — target-verified WB comparison
- PMC11435019 — target-verified WB comparison
- PMC13171308 — target-verified WB comparison