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- Table of Contents
WT1 / Wilms tumor protein · Western blot design guide
Design a Western Blot for WT1
Real validated WT1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-WT1 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
WT1 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 | ~49.2 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Breast (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 | Ubl conjugation | |
| Caveat | Isoform-specific interpretation | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 8 isoform(s) |
Section 1
Real Curated WT1 Western Blot Protocols
The M00199-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | K562 cell lysate (catalog M00199-1) |
| 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 | M00199-1; 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 WT1 Western Blot Band Size?
WT1 is predicted at 49.2 kDa; homodimerization and isoforms could affect the pattern, but no empirical migration difference is demonstrated.
| Band near 49.2 kDa | consistent with the predicted WT1 monomer; confirm identity with controls |
| Band near twice the monomer size | could reflect WT1 homodimerization |
| Several bands at different positions | could reflect WT1 isoforms; their migration differences are unestablished |
| Band in a nuclear fraction | consistent with nuclear WT1, which can also shuttle to the cytoplasm |
💡Expected WT1 appearanceWT1 has a predicted mass of 49.2 kDa, but no empirical band size is supplied; confirm any candidate band with antibody and sample controls.
| Predicted WT1 mass | places the monomer near 49.2 kDa before any demonstrated migration effect |
| WT1 homodimer | could produce a band near twice the monomer size if the complex persists during electrophoresis |
| Splice isoforms 1, 2, 3, and 4 | may differ in apparent size; their relative sizes and migration are unspecified |
| Splice isoforms 6, 7, 8, and 9 | may differ in apparent size; their relative sizes and migration are unspecified |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | nuclear WT1 may be poorly recovered in the preparation | check nuclear extraction and include a WT1-positive lysate |
| Band higher than expected | WT1 homodimer may persist during electrophoresis | compare reducing and nonreducing conditions and verify band identity |
| Band lower than expected | an isoform could differ in size, but its migration is unspecified | verify the band with an independent WT1 antibody or isoform-aware control |
| Multiple bands | WT1 has eight named isoforms, though distinct bands are unproven | compare antibody epitopes and use WT1 depletion to identify specific bands |
| Weak or no signal | WT1 distribution between nucleus and cytoplasm may limit signal in the sampled fraction | check both fractions and include a WT1-positive control |
Experiment evidence
Western blot images showing WT1 band
M00199-1 · WT1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for WT1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for WT1 in Western blot, you can use breast tissue, which has high WT1 expression in HPA.
Positive control: Breast (IHC candidate; verify WB)
Negative control: Adrenal gland (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: WT1 is mainly nuclear, so nuclear-enriched lysate may improve detection.
HPA tissue expression evidence for WT1
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 |
|---|---|---|---|---|
| Breast | myoepithelial cells | High | Protein (IHC) | HPA → |
| Endometrium | endothelial cells | High | Protein (IHC) | HPA → |
| Fallopian tube | glandular cells | High | Protein (IHC) | HPA → |
| Kidney | cells in glomeruli | High | Protein (IHC) | HPA → |
| Testis | sertoli cells | High | 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 → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced WT1 Western Blot Tips
Deeper troubleshooting and optimisation questions for WT1, answered from its protein features.
How should WT1 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.
Which WT1 isoforms could produce different bands?
Isoforms · The listed isoforms are 1, 2, 3, 4, 6, 7, 8, and 9. Their annotated differences include changes at UniProt positions 1–144, 145–147, 250–266, and 408–410. Compare the antibody epitope with these regions before interpreting multiple bands as isoforms.
Yes. UniProt positions 1–144 are missing in isoforms 6 and 9, while isoforms 7 and 8 have an alternative sequence beginning at position 1. Check whether the antibody epitope is retained in the isoform you intend to detect.
Isoforms 2, 4, and 6 lack UniProt positions 408–410, the KTS motif. The supplied localization note says isoforms lacking KTS have a diffuse nuclear location. A three-residue difference alone does not establish that separate bands will resolve.
Yes. UniProt positions 1–144 are missing in isoforms 6 and 9, while isoforms 7 and 8 have an alternative sequence beginning at position 1. Check whether the antibody epitope is retained in the isoform you intend to detect.
Isoforms 2, 4, and 6 lack UniProt positions 408–410, the KTS motif. The supplied localization note says isoforms lacking KTS have a diffuse nuclear location. A three-residue difference alone does not establish that separate bands will resolve.
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 WT1?
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.
How should transfer be checked for WT1?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M00199-1 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 WT1 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.
How should I interpret a band away from 49.2 kDa?
Interpretation · 49.2 kDa is the predicted mass, and no observed band position is supplied. WT1 has eight listed isoforms with sequence differences that could affect apparent size. A band shift cannot be assigned to a specific isoform from mass alone.
Use an antibody epitope shared by the isoforms you want to quantify, or report which isoforms the assay detects. Differences at UniProt positions 1–144, 250–266, and 408–410 can change epitope coverage, so signal intensity may not represent total WT1 equally across isoforms.
WT1 is annotated in the nucleus, nucleolus, and cytoplasm and shuttles between nucleus and cytoplasm. Isoforms lacking KTS have a diffuse nuclear location. Interpret fraction signals with these localization features and the antibody’s isoform coverage in mind.
First compare antibody epitope coverage with the annotated isoform changes, especially the large deletion at UniProt positions 1–144 in isoforms 6 and 9 and the deletion at 250–266 in isoforms 2, 3, 8, and 9. The supplied features do not establish the identity of any observed band.
Use an antibody epitope shared by the isoforms you want to quantify, or report which isoforms the assay detects. Differences at UniProt positions 1–144, 250–266, and 408–410 can change epitope coverage, so signal intensity may not represent total WT1 equally across isoforms.
WT1 is annotated in the nucleus, nucleolus, and cytoplasm and shuttles between nucleus and cytoplasm. Isoforms lacking KTS have a diffuse nuclear location. Interpret fraction signals with these localization features and the antibody’s isoform coverage in mind.
First compare antibody epitope coverage with the annotated isoform changes, especially the large deletion at UniProt positions 1–144 in isoforms 6 and 9 and the deletion at 250–266 in isoforms 2, 3, 8, and 9. The supplied features do not establish the identity of any observed band.
Boster reagents
WT1 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-Wilms Tumor Protein WT1 Rabbit Monoclonal Antibody
Cat # M00199-1
M00199-1 is a rabbit monoclonal anti-WT1 antibody listed for human and mouse reactivity. Its Western blot image shows WT1 expression in K562 cell lysate; the supplied evidence does not show a mouse sample.
Which to pick: Only M00199-1 is listed. Choose it if its listed human or mouse reactivity fits your sample, while noting that the supplied Western blot example uses K562 cell lysate.
Source: BosterBio WT1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P19544.
- Human Protein Atlas. WT1 tissue expression.
- PMC11166697 — target-verified WB comparison
- PMC4773705 — target-verified WB comparison
- PMC4421988 — target-verified WB comparison