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- Table of Contents
YTHDF1 / YTH domain-containing family protein 1 · Western blot design guide
Design a Western Blot for YTHDF1
Real validated YTHDF1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-YTHDF1 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
YTHDF1 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 | ~60.9 kDa | |
| Observed band | ~65–67 kDa | |
| Gel | 8% (catalog A09853-2) | |
| 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 | 2 isoform(s) |
Section 1
Real Curated YTHDF1 Western Blot Protocols
The A09853-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human A549, human 293T, human Hela (catalog A09853-2) |
| Gel % | 8% (catalog A09853-2) |
| Load | 30 ug; reducing conditions (catalog A09853-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A09853-2) |
| Membrane | nitrocellulose membrane (catalog A09853-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A09853-2) |
| Primary antibody | A09853-2 · 1:1000 (catalog A09853-2) |
| Primary incubation | overnight at 4°C (catalog A09853-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A09853-2) |
| Secondary incubation | 1.5 hour at RT (catalog A09853-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A09853-2) |
| Detection | ECL (catalog A09853-2) |
Section 2
What Is the Expected YTHDF1 Western Blot Band Size?
YTHDF1 is predicted at 60.9 kDa and observed at ~65–67 kDa; the cause of the difference is not established.
| Band at ~65–67 kDa | Empirically observed YTHDF1 band in whole-cell lysates; confirm identity with controls |
| Band near 60.9 kDa | Consistent with the predicted YTHDF1 mass; confirm identity with controls |
| Two bands at different positions | Isoforms 1 and 2 are possible contributors, but distinct migration is unestablished |
| Band in a cytoplasmic fraction | Consistent with YTHDF1 cytoplasmic localization |
💡Expected YTHDF1 appearanceYTHDF1 has a predicted mass of 60.9 kDa and an observed whole-cell band at ~65–67 kDa; the cause of the difference is unestablished, so confirm identity with ordinary band controls.
| Predicted YTHDF1 mass | 60.9 kDa is the sequence-based reference; the observed band is ~65–67 kDa |
| Isoform 1 | Its size relative to isoform 2 is not supplied |
| Isoform 2 | Its size relative to isoform 1 is not supplied |
| Alternative splicing | Could alter protein size, but distinct band positions are unestablished |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Poor recovery of cytoplasmic YTHDF1 | Check cytoplasmic protein recovery, loading, and a positive-control lysate |
| Band higher than expected | The observed ~65–67 kDa band exceeds the 60.9 kDa prediction; the reason is unestablished | Compare with a validated positive control and verify antibody specificity |
| Band lower than expected | Possible isoform difference or protein breakdown; neither band position is established | Check sample integrity and confirm identity with an independent antibody or depletion control |
| Multiple bands | Isoforms 1 and 2 are documented, but their migration is unknown | Use an isoform-aware control and verify band identity |
| Weak or no signal | Low recovery of cytoplasmic YTHDF1 | Check extraction, loading, transfer, and antibody performance with a positive control |
Experiment evidence
Western blot images showing YTHDF1 band
A09853-2 · YTHDF1
A09853-2 · YTHDF1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for YTHDF1 Western blot
🧪For positive controls for YTHDF1 in Western blot, you can use a positive sample once HPA evidence identifies one.
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: HPA data are unavailable, so a validated positive sample and a clean negative tissue cannot be selected.
HPA tissue expression evidence for YTHDF1
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 YTHDF1 Western Blot Tips
Deeper troubleshooting and optimisation questions for YTHDF1, answered from its protein features.
How should YTHDF1 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 YTHDF1 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. Relative to the canonical sequence, isoform 2 lacks residues 1–190 and 383–559 and replaces residues 191–260. These substantial sequence differences could yield distinct bands. Check whether the antibody recognizes a region retained in isoform 2 before interpreting band patterns.
Isoform 2 lacks canonical residues 1–190 and 383–559, and its residues corresponding to 191–260 differ. An antibody targeting one of those canonical regions may fail to recognize isoform 2. Check the immunogen or epitope location before treating an absent second band as evidence that isoform 2 is absent.
Isoform 2 lacks canonical residues 1–190 and 383–559, and its residues corresponding to 191–260 differ. An antibody targeting one of those canonical regions may fail to recognize isoform 2. Check the immunogen or epitope location before treating an absent second band as evidence that isoform 2 is absent.
Which YTHDF1 modifications matter when interpreting bands?
PTM · UniProt lists N-acetylserine at position 2 and phosphoserine at position 182, using canonical sequence coordinates. Both positions fall within the region missing from isoform 2. Their presence does not establish a detectable band shift; compare appropriately controlled samples before assigning a shifted band to a modification.
Does this guide establish induction of YTHDF1?
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 YTHDF1?
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 A09853-2 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 YTHDF1 bands be quantified?
Quantitation · Choose a consistently detected band and verify which isoform the antibody recognizes. UniProt lists two isoforms with substantial sequence differences, so combining multiple bands without identifying them could obscure changes in either isoform. Keep sample preparation consistent because YTHDF1 is reported in cytoplasm, P-bodies, and stress granules.
Why might YTHDF1 appear near 65–67 kDa instead of 60.9 kDa?
Interpretation · The supplied predicted mass is 60.9 kDa, while the observed band is approximately 65–67 kDa. UniProt lists N-acetylserine at position 2 and phosphoserine at position 182, but these features alone do not explain the difference or establish a visible shift. Confirm band identity with a YTHDF1-specific control.
UniProt places YTHDF1 in the cytoplasm, P-bodies, and stress granules. Use comparable sample preparation across conditions, especially when comparing cellular fractions, because changes in recovery from these compartments could affect measured signal.
Check antibody specificity and its target region against the two listed isoforms. Isoform 2 lacks canonical residues 1–190 and 383–559 and differs at 191–260. UniProt also lists modifications at canonical positions 2 and 182, but those entries alone cannot identify an unexpected band or establish a visible shift.
UniProt places YTHDF1 in the cytoplasm, P-bodies, and stress granules. Use comparable sample preparation across conditions, especially when comparing cellular fractions, because changes in recovery from these compartments could affect measured signal.
Check antibody specificity and its target region against the two listed isoforms. Isoform 2 lacks canonical residues 1–190 and 383–559 and differs at 191–260. UniProt also lists modifications at canonical positions 2 and 182, but those entries alone cannot identify an unexpected band or establish a visible shift.
Boster reagents
YTHDF1 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-YTHDF1 Antibody Picoband®
Cat # A09853-2
The catalog reports one anti-YTHDF1 Western blot antibody, A09853-2, with reported human, mouse, and rat reactivity. Its WB image includes cell and tissue lysates from all three species; the reported band is approximately 67 kDa versus an expected 61 kDa. No orthogonal validation is supplied.
Which to pick: A09853-2 is the only listed option and has a WB image. The caption reports 30 µg per lane under reducing conditions and a 1:1000 primary dilution. Check the observed 67 kDa band against your sample and expected size.
Source: BosterBio YTHDF1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9BYJ9.
- Human Protein Atlas. YTHDF1 tissue expression.
- PMC8594712 — target-verified WB comparison
- PMC6226095 — target-verified WB comparison
- PMC8867832 — target-verified WB comparison