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- Table of Contents
YTHDF3 / YTH domain-containing family protein 3 · Western blot design guide
Design a Western Blot for YTHDF3
Real validated YTHDF3 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-YTHDF3 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
YTHDF3 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 | ~63.9 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 | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated YTHDF3 Western Blot Protocols
The A14443 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | extracts of Mouse liver, (catalog A14443) |
| 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 | Blocking buffer: 3% nonfat dry milk in TBST (catalog A14443) |
| Primary antibody | A14443 · 1:1000 (catalog A14443) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Goat Anti-Rabbit IgG, 1:10000 (catalog A14443) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL (catalog A14443) |
Section 2
What Is the Expected YTHDF3 Western Blot Band Size?
YTHDF3 is predicted at 63.9 kDa; Ser2 acetylation and Ser23 phosphorylation are annotated, but their effects on migration are not demonstrated.
| Band near 63.9 kDa | Consistent with the predicted full-length YTHDF3 mass; confirm identity with controls. |
| Closely spaced bands near 63.9 kDa | Ser23 phosphorylation is a possible contributor, but distinct migration is unproven. |
| Band near 63.9 kDa without a visible shift | Ser2 acetylation need not produce a resolvable shift. |
| Band in a cytosolic fraction | Consistent with YTHDF3 localization in the cytosol. |
💡Expected YTHDF3 appearanceFull-length YTHDF3 has a predicted mass of 63.9 kDa, with no empirical band size supplied; use band-identity controls because its annotated modifications do not establish a visible shift.
| UniProt predicted mass | Places the full-length protein near 63.9 kDa. |
| 585-residue sequence | Defines the full-length protein underlying the predicted mass. |
| Ser2 N-acetylserine | Adds a small modification without an established visible shift. |
| Ser23 phosphoserine | May affect mobility, but no distinct band position is established. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Low YTHDF3 signal or poor recovery from its cytosolic compartment. | Check loading and detection with a cytosolic positive control. |
| Band higher than expected | Band identity or a mobility effect is unverified; Ser23 phosphorylation is annotated. | Compare phosphatase-treated samples and confirm identity with an independent antibody or knockdown. |
| Band lower than expected | Possible degradation; no cleavage feature is annotated. | Prepare fresh lysate with protease inhibitors and confirm identity by knockdown. |
| Multiple bands | Phosphorylation is possible, but separate YTHDF3 bands are not established. | Compare phosphatase treatment and knockdown to identify YTHDF3-dependent bands. |
| Weak or no signal | Insufficient cytosolic protein or detection sensitivity. | Verify protein loading and use a YTHDF3-positive cytosolic sample. |
| Fragments below expected size | Possible sample degradation rather than annotated processing. | Prepare fresh lysate with protease inhibitors and check whether fragments track with YTHDF3 knockdown. |
Experiment evidence
Western blot images showing YTHDF3 band
A14443 · YTHDF3
A14443 · YTHDF3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for YTHDF3 Western blot
🧪For positive controls for YTHDF3 in Western blot, you can use no HPA-selected sample because no positive 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: Cytosolic localization supports lysate testing, but HPA data are unavailable to identify tissue controls.
HPA tissue expression evidence for YTHDF3
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 YTHDF3 Western Blot Tips
Deeper troubleshooting and optimisation questions for YTHDF3, answered from its protein features.
Where should the YTHDF3 band appear?
Band shift · The predicted mass is 63.9 kDa. No observed band position is supplied, so use 63.9 kDa as a reference rather than an exact migration target.
Could multiple isoforms explain extra YTHDF3 bands?
Isoforms · Only one isoform is supplied, with no alternative sequence. The supplied features therefore provide no basis for assigning extra bands to YTHDF3 isoforms.
Does YTHDF3 phosphorylation predict a visible band shift?
PTM · UniProt lists phosphoserine at position 23. This annotation alone does not establish a visible shift or explain a difference between apparent and predicted mass. Keep the UniProt numbering explicit when comparing site-specific reagents or reports.
UniProt lists N-acetylserine at position 2. This is UniProt sequence numbering; check numbering conventions before comparing it with antibody or paper annotations. Its presence alone does not predict a detectable shift.
UniProt lists N-acetylserine at position 2. This is UniProt sequence numbering; check numbering conventions before comparing it with antibody or paper annotations. Its presence alone does not predict a detectable shift.
Does this guide establish induction of YTHDF3?
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 YTHDF3?
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 A14443 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 YTHDF3 Western blot signals be quantified?
Quantitation · Use consistent cytoplasmic preparation across samples. Because YTHDF3 is annotated in the cytosol, P-bodies, and stress granules, a change in one fraction alone does not establish a change in total YTHDF3.
Which cell fraction is relevant for detecting YTHDF3?
Interpretation · YTHDF3 is annotated in the cytosol, P-bodies, and stress granules. Include the cytoplasmic fraction when assessing detection, and compare equivalent fractions across samples.
Compare them with the 63.9 kDa predicted mass, but do not assign their identity from migration alone. The listed modifications and interactions with CNOT1, YTHDF1, YTHDF2, and PAN3 do not establish the cause of an extra band.
Compare them with the 63.9 kDa predicted mass, but do not assign their identity from migration alone. The listed modifications and interactions with CNOT1, YTHDF1, YTHDF2, and PAN3 do not establish the cause of an extra band.
Boster reagents
YTHDF3 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-YTHDF3 Antibody
Cat # A14443
A14443 is listed as reactive with human, mouse, and rat YTHDF3. The supplied Western blot caption documents mouse liver extract tested at 1:1000 antibody dilution; it provides no human or rat blot examples.
Which to pick: A14443 is the only listed option and has a Western blot image from mouse liver extract. For other sample types, the supplied evidence does not show a blot.
Source: BosterBio YTHDF3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q7Z739.
- Human Protein Atlas. YTHDF3 tissue expression.
- PMC13249798 — target-verified WB comparison
- PMC13576909 — target-verified WB comparison
- PMC11449838 — target-verified WB comparison