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- Table of Contents
YWHAH / 14-3-3 protein eta · Western blot design guide
Design a Western Blot for YWHAH
Source-linked YWHAH Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-YWHAH 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
YWHAH 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 | ~28.2 kDa | |
| Observed band | ~28 kDa | |
| Gel | 5–20% (catalog A04219-2) | |
| Positive control | Cerebellum (IHC candidate; verify WB) +4 more | |
| Negative control | Duodenum (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 Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked YWHAH Western Blot Protocol Options
The A04219-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Jurkat, human Hela, monkey COS-7, human 293T, rat brain, rat PC-12, mouse brain, mouse NIH/3T3 (catalog A04219-2) |
| Gel % | 5–20% (catalog A04219-2) |
| Load | 30 ug; reducing conditions (catalog A04219-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A04219-2) |
| Membrane | nitrocellulose membrane (catalog A04219-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A04219-2) |
| Primary antibody | A04219-2 · 0.5 μg/mL (catalog A04219-2) |
| Primary incubation | overnight at 4°C (catalog A04219-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A04219-2) |
| Secondary incubation | 1.5 hour at RT (catalog A04219-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A04219-2) |
| Detection | ECL (catalog A04219-2) |
Section 2
What Is the Expected YWHAH Western Blot Band Size?
YWHAH is predicted at 28.2 kDa and observed at ~28 kDa; its listed modifications have no demonstrated visible migration effect.
| Dominant band at ~28 kDa | Matches the empirical YWHAH band and its 28.2 kDa predicted mass |
| Band near ~56 kDa under conditions that preserve dimers | Could reflect a homodimer, reported by similarity; confirm its identity |
| Closely spaced bands near ~28 kDa | Could reflect phosphorylation at Ser25 or Ser59, but mobility alone cannot establish this |
| Single band near ~28 kDa without a resolved doublet | N-acetylglycine at residue 2 and any phosphorylation need not produce separate visible bands |
💡Expected YWHAH appearanceYWHAH has a predicted mass of 28.2 kDa and an empirical band at ~28 kDa in reducing whole-cell lysates; confirm band identity with antibody and sample controls.
| Predicted YWHAH mass | 28.2 kDa predicts a band close to the empirical ~28 kDa band |
| Homodimer by similarity | Could appear near twice the monomer mass if the dimer survives sample preparation |
| Phosphoserine at Ser25 | May affect mobility, but no visible shift is established |
| Phosphoserine at Ser59 | May affect mobility, but no visible shift is established |
| N-acetylglycine at residue 2 | Does not establish a resolvable size change |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | A retained homodimer is possible, though not established by band position | Compare denaturing and reducing preparation, then verify band identity |
| Multiple bands | Phosphorylation at Ser25 or Ser59 is possible, but the bands are unassigned | Compare phosphatase-treated and untreated samples with band-identity controls |
| Band lower than expected | The listed features do not establish a smaller mature form | Check sample integrity and verify the band with an independent antibody |
| Fragments below expected size | Sample degradation or nonspecific antibody binding may produce smaller bands | Prepare fresh lysate with protease inhibitors and verify band identity |
| Weak or no signal | Low target recovery or detection sensitivity may obscure the ~28 kDa band | Check loading, transfer, antibody conditions and a positive-control lysate |
Experiment evidence
Western blot images showing YWHAH band
A04219-2 · YWHAH
A04219 · YWHAH
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for YWHAH Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for YWHAH in Western blot, you can use cerebellum lysate, which has high HPA expression.
Positive control: Cerebellum (IHC candidate; verify WB)
Negative control: Duodenum (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: HPA reports YWHAH as not detected in duodenum, making it a candidate negative tissue control.
HPA tissue expression evidence for YWHAH
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 |
|---|---|---|---|---|
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuropil | High | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Duodenum | glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Seminal vesicle | glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced YWHAH Western Blot Tips
Deeper troubleshooting and optimisation questions for YWHAH, answered from its protein features.
How should YWHAH 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 a second band be a YWHAH isoform?
Isoforms · The supplied record lists one isoform and no alternative sequence. It provides no isoform-based explanation for a second band; confirm that band's identity before assigning it to YWHAH.
Which phosphorylation sites matter when interpreting YWHAH bands?
PTM · UniProt lists phosphoserine at positions 25 and 59. These are UniProt coordinates; antibody or paper numbering may differ. Their presence does not show that phosphorylation causes a visible band shift.
UniProt lists N-acetylglycine at position 2. This modification alone does not establish an apparent mass difference. Check the expected ~28 kDa band before attributing another band to acetylation.
UniProt lists N-acetylglycine at position 2. This modification alone does not establish an apparent mass difference. Check the expected ~28 kDa band before attributing another band to acetylation.
Does this guide establish induction of YWHAH?
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 YWHAH?
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 A04219-2 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.
Which band should be quantified for YWHAH?
Quantitation · Quantify the reported ~28 kDa band consistently; it aligns with the 28.2 kDa predicted mass. Keep other bands separate unless their YWHAH identity is established.
Should YWHAH migrate at its predicted mass?
Interpretation · YWHAH is predicted at 28.2 kDa, consistent with the reported apparent band near 28 kDa. Use that band as the initial target; the listed modifications alone do not establish a visible shift.
YWHAH is annotated as a homodimer by similarity. That annotation makes a dimer worth considering, but does not establish that a higher band is a dimer on your blot. Verify the band's identity before labeling it.
Start with the ~28 kDa expected band. UniProt lists phosphorylation at positions 25 and 59, N-acetylglycine at position 2, and homodimerization by similarity, but none establishes the identity or migration of an unexpected band. Confirm its identity before assigning a cause.
YWHAH is annotated as a homodimer by similarity. That annotation makes a dimer worth considering, but does not establish that a higher band is a dimer on your blot. Verify the band's identity before labeling it.
Start with the ~28 kDa expected band. UniProt lists phosphorylation at positions 25 and 59, N-acetylglycine at position 2, and homodimerization by similarity, but none establishes the identity or migration of an unexpected band. Confirm its identity before assigning a cause.
Boster reagents
YWHAH 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-YWHAH Antibody Picoband®
Cat # A04219-2
Real WB data
Anti-14-3-3 eta YWHAH Antibody
Cat # A04219
The catalog reports two anti-YWHAH antibodies with WB images. A04219-2 shows an approximately 28 kDa band in the illustrated human, monkey, rat, and mouse lysates. A04219 shows Jurkat lysate with a peptide-blocked lane; its image does not document other specimens.
Which to pick: Pick A04219-2 for a WB example spanning human, monkey, rat, and mouse specimens. A04219 lists human, mouse, and rat reactivity, but its shown WB uses Jurkat cells. Both have WB images; only A04219-2 shows the broader specimen set.
Source: BosterBio YWHAH gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q04917.
- Human Protein Atlas. YWHAH tissue expression.
- PMC11751264 — target-verified WB comparison
- PMC11262655 — target-verified WB comparison
- PMC11035551 — target-verified WB comparison