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UBE2D3 / Ubiquitin-conjugating enzyme E2 D3 · Western blot design guide
Design a Western Blot for UBE2D3
Source-linked UBE2D3 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-UBE2D3 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
UBE2D3 Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~16.7 kDa | |
| Gel | 15% (standard starting point) | |
| Positive control | Adipose tissue (IHC candidate; verify WB) +4 more | |
| Negative control | Adrenal gland (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked UBE2D3 Western Blot Protocol Options
The A03923 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | extracts of various cell lines, (catalog A03923) |
| Gel % | 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 | 0.2 µm PVDF (standard starting point) |
| Blocking | Blocking buffer: 3% nonfat dry milk in TBST (catalog A03923) |
| Primary antibody | A03923 · 1:1000 (catalog A03923) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Goat Anti-Rabbit IgG, 1:10000 (catalog A03923) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL (catalog A03923) |
Section 2
What Is the Expected UBE2D3 Western Blot Band Size?
UBE2D3 is predicted at 16.7 kDa; isoforms and an intrachain disulfide could affect migration, but no empirical band or feature-dependent shift is demonstrated.
| Band near 16.7 kDa | consistent with the predicted UBE2D3 mass; identity requires confirmation |
| Additional band at another size | could reflect one of isoforms 1, 2, or 3; their migration is unknown |
| Mobility changes after reduction | could reflect the intrachain disulfide between residues 21 and 107 |
| Weak band in a soluble fraction | could reflect UBE2D3 association with cell or endosome membranes |
💡Expected UBE2D3 appearanceUBE2D3 has a predicted mass of 16.7 kDa, but no empirical band size is supplied; confirm any candidate band with an identity control because isoform migration and disulfide effects are unestablished.
| Predicted UBE2D3 mass | 16.7 kDa is the sequence-based reference, not a measured band position |
| Isoform 1 | its size relative to isoforms 2 and 3 is not supplied |
| Isoform 2 | its size relative to isoforms 1 and 3 is not supplied |
| Isoform 3 | its size relative to isoforms 1 and 2 is not supplied |
| Intrachain disulfide between residues 21 and 107 | may affect migration under nonreducing conditions; no shift is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | UBE2D3 may be poorly recovered from membrane-associated material | check membrane fraction recovery and use a validated positive-control lysate |
| Band higher than expected | the band's identity or migration is unestablished | compare reducing and nonreducing lanes and confirm identity by UBE2D3 depletion |
| Band lower than expected | an isoform or fragment is possible, but its mass is unknown | confirm identity by UBE2D3 depletion and compare antibodies to different epitopes |
| Multiple bands | isoforms 1, 2, and 3 or unrelated antibody binding may contribute | use UBE2D3 depletion to identify specific bands |
| Weak or no signal | membrane-associated UBE2D3 may be underrepresented in the sample | check extraction and loading with a validated positive control |
Experiment evidence
Western blot images showing UBE2D3 band
A03923 · UBE2D3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for UBE2D3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for UBE2D3 in Western blot, you can use adipose tissue lysate.
Positive control: Adipose tissue (IHC candidate; verify WB)
Negative control: Adrenal gland (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: UBE2D3 is membrane-associated, so prepare lysates that retain membrane-associated proteins.
HPA tissue expression evidence for UBE2D3
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 |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | glandular cells | Medium | Protein (IHC) | HPA → |
| Colon | glandular cells | Medium | Protein (IHC) | HPA → |
| Fallopian tube | glandular cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced UBE2D3 Western Blot Tips
Deeper troubleshooting and optimisation questions for UBE2D3, answered from its protein features.
Where should the UBE2D3 band appear?
Band shift · The predicted mass is 16.7 kDa. No observed band position is supplied, so use 16.7 kDa as a starting point rather than an exact apparent mass. The listed features alone cannot establish a band shift.
Could UBE2D3 isoforms produce nearby bands?
Isoforms · Three isoforms are listed. Relative to the 147-residue canonical sequence, isoform 3 changes residues 1–8 and isoform 2 changes residues 134–147, with small sequence-length differences. Nearby bands are possible, but these features do not establish that the isoforms resolve separately. Check whether the antibody recognizes the relevant sequence.
Do listed modifications explain a shifted UBE2D3 band?
PTM · UBE2D3 is labeled a phosphoprotein and has one listed disulfide bond, but this record supplies no modified-residue coordinates or glycosylation sites. These annotations do not establish a visible shift or explain a measured mass difference.
Does this guide establish induction of UBE2D3?
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 UBE2D3 Western blot?
Transfer · UBE2D3 is a small, 16.7 kDa protein. Choose transfer conditions and a membrane that retain small proteins, then check transfer efficiency around 17 kDa. The supplied features do not specify a particular transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A03923 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 UBE2D3 be quantified across fractions?
Quantitation · UBE2D3 is annotated as a peripheral protein at the cell and endosome membranes. Compare like fractions across samples, keep loading and exposure consistent, and verify that the detected band is within the assay’s linear range. A change in one fraction may reflect redistribution rather than a change in total UBE2D3.
How should unexpected UBE2D3 bands be interpreted?
Interpretation · First compare them with the expected 16.7 kDa region and consider the three annotated isoforms. UBE2D3 interacts with the SCF complex, with stronger interaction when Cullin is neddylated, and with DAPK3; those interactions alone do not identify an extra band. Check antibody specificity before assigning it to an isoform or modification.
Boster reagents
UBE2D3 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-UBE2D3 Antibody
Cat # A03923
A03923 is listed for Western blot with reported human, mouse, and rat reactivity. Its WB image shows extracts from various cell lines tested at 1:1000 dilution and 25 µg per lane. The caption does not identify the cell lines.
Which to pick: A03923 is the only listed UBE2D3 antibody and has a WB image. Its reported 1:1000 primary dilution is a starting point, but the image caption does not identify which cell lines were tested.
Source: BosterBio UBE2D3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P61077.
- Human Protein Atlas. UBE2D3 tissue expression.
- PMC4911883 — target-verified WB comparison
- PMC13285137 — target-verified WB comparison
- PMC10909922 — target-verified WB comparison