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- Table of Contents
WTAP / Pre-mRNA-splicing regulator WTAP · Western blot design guide
Design a Western Blot for WTAP
Real validated WTAP Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-WTAP 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
WTAP 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 | ~44.2 kDa | |
| Observed band | ~50 kDa | |
| Gel | 8% (catalog A04296-2) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Heart muscle (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated WTAP Western Blot Protocols
The A04296-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human 293T, human THP-1 (catalog A04296-2) |
| Gel % | 8% (catalog A04296-2) |
| Load | 30 ug; reducing conditions (catalog A04296-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A04296-2) |
| Membrane | nitrocellulose membrane (catalog A04296-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A04296-2) |
| Primary antibody | A04296-2 · 1:1000 (catalog A04296-2) |
| Primary incubation | overnight at 4°C (catalog A04296-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A04296-2) |
| Secondary incubation | 1.5 hour at RT (catalog A04296-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A04296-2) |
| Detection | ECL (catalog A04296-2) |
Section 2
What Is the Expected WTAP Western Blot Band Size?
WTAP is predicted at 44.2 kDa and observed near 50 kDa; the cause of this difference is not established by the supplied evidence.
| Band at ~50 kDa | Empirical WTAP band in reducing whole-cell lysates; confirm identity with controls |
| Band near 44.2 kDa | Near the predicted WTAP mass, but band identity needs confirmation |
| Two bands at different positions | Could reflect WTAP isoforms 1 and 2; distinct migration is unverified |
| Faint band in a cytoplasmic fraction | Consistent with WTAP being mainly nuclear |
💡Expected WTAP appearanceWTAP has a predicted mass of 44.2 kDa, while antibody QC shows a ~50 kDa band in reducing whole-cell lysates; the difference is unexplained, so confirm identity with ordinary controls.
| Predicted WTAP mass | 44.2 kDa from the UniProt sequence; observed migration is ~50 kDa |
| Alternative splicing | Produces isoforms 1 and 2, whose relative apparent sizes are unspecified |
| WTAP isoform 1 | Its apparent size relative to isoform 2 is not supplied |
| WTAP isoform 2 | Its apparent size relative to isoform 1 is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | WTAP is mainly nuclear or its signal is below detection | Check a validated positive lysate and a nuclear fraction |
| Band higher than expected | The ~50 kDa empirical band exceeds the 44.2 kDa prediction for an undetermined reason | Compare with the validated ~50 kDa band and confirm identity by WTAP depletion |
| Band lower than expected | An alternative isoform or sample degradation is possible | Check sample integrity and confirm the band by WTAP depletion |
| Multiple bands | Isoforms 1 and 2 may contribute, but their migration is unspecified | Use WTAP depletion and isoform-aware validation to identify the bands |
| Weak or no signal | Limited WTAP signal in the tested fraction is possible | Check a validated positive lysate and enrich the nuclear fraction |
| Fragments below expected size | Sample degradation is possible; no WTAP cleavage feature is supplied | Prepare fresh lysate with protease inhibitors and confirm band identity |
Experiment evidence
Western blot images showing WTAP band
A04296-2 · WTAP
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for WTAP Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for WTAP in Western blot, you can use adrenal gland tissue, which HPA rates High.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Heart muscle (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside.
⚠️Feasibility: WTAP is mainly nuclear, so nuclear-enriched lysate may give a clearer positive signal.
HPA tissue expression evidence for WTAP
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Low | Protein (IHC) | HPA → |
| Salivary gland | glandular cells | Low | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Low | Protein (IHC) | HPA → |
| Spleen | cells in white pulp | Low | Protein (IHC) | HPA → |
Section 3
Advanced WTAP Western Blot Tips
Deeper troubleshooting and optimisation questions for WTAP, answered from its protein features.
How should WTAP 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 WTAP isoforms produce different bands?
Isoforms · UniProt lists two isoforms. Relative to isoform 1, isoform 2 has S151R and lacks residues 152–396, using UniProt canonical coordinates. A lower band could be consistent with isoform 2, but band position alone cannot confirm its identity. Check whether the antibody epitope is retained in isoform 2.
Which WTAP modifications matter when interpreting bands?
PTM · UniProt canonical coordinates list N-acetylmethionine at 1; phosphoserine at 14, 305, 306, 341, and 388; and phosphothreonine at 350. Isoform 2 lacks the listed sites from 305 onward. These annotations do not establish a visible band shift.
Does this guide establish induction of WTAP?
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 WTAP?
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 A04296-2 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 WTAP localization affect quantitation?
Quantitation · WTAP is mainly nuclear, in speckles and nucleoplasm, with some cytoplasmic fraction. Keep the sample fraction consistent across lanes and comparisons. A nuclear extract and a whole-cell lysate may yield different WTAP amounts even when starting material is comparable.
Why does WTAP appear near 50 kDa instead of 44.2 kDa?
Interpretation · 44.2 kDa is the predicted mass, while ~50 kDa is the observed apparent band. WTAP has annotated modifications, but their presence alone does not establish the cause of this difference. Use the observed band position alongside antibody specificity controls when identifying WTAP.
Consider isoform 2, which lacks UniProt canonical residues 152–396. An antibody recognizing that deleted region would not detect intact isoform 2. Compare the band with the antibody epitope and the expected isoform sequence before assigning it to WTAP.
The supplied features list no signal peptide, propeptide, or glycosylation sites. They therefore provide no basis for assigning an additional band to cleavage of those peptides or glycosylation. The listed isoform difference and modifications offer possibilities to investigate, but neither identifies an unexpected band by itself.
Consider isoform 2, which lacks UniProt canonical residues 152–396. An antibody recognizing that deleted region would not detect intact isoform 2. Compare the band with the antibody epitope and the expected isoform sequence before assigning it to WTAP.
The supplied features list no signal peptide, propeptide, or glycosylation sites. They therefore provide no basis for assigning an additional band to cleavage of those peptides or glycosylation. The listed isoform difference and modifications offer possibilities to investigate, but neither identifies an unexpected band by itself.
Boster reagents
WTAP 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-WTAP Antibody
Cat # A04296-2
A04296-2 is a rabbit polyclonal anti-WTAP antibody listed for human, mouse, and rat. Its WB caption reports a band near 50 kDa, versus 44 kDa expected, in the tested cell lysates. No publication or independent validation evidence is supplied.
Which to pick: A04296-2 is the only listed option and has a WB image. The caption reports HeLa, 293T, THP-1, K562, PC-12, and NRK lysates tested at 1:1000 primary dilution. Match your sample and conditions to these reported examples when planning a blot.
Source: BosterBio WTAP gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q15007.
- Human Protein Atlas. WTAP tissue expression.
- PMC11799344 — target-verified WB comparison
- PMC8083052 — target-verified WB comparison
- PMC5827993 — target-verified WB comparison