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PSMD14 / Ubiquitin C-terminal hydrolase PSMD14 · Western blot design guide
Design a Western Blot for PSMD14
Source-linked PSMD14 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PSMD14 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
PSMD14 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 | ~34.6 kDa | |
| Observed band | ~35 kDa | |
| Gel | 10% (catalog M06584) | |
| Positive control | Adipose tissue (IHC candidate; verify WB) +4 more | |
| Negative control | Fallopian tube (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked PSMD14 Western Blot Protocol Options
The M06584 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human 293T, human Hela, human Jurkat, human HT1080, rat heart, rat skeletal muscle, mouse heart, mouse skeletal muscle (catalog M06584) |
| Gel % | 10% (catalog M06584) |
| Load | 30 ug; reducing conditions (catalog M06584) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M06584) |
| Membrane | nitrocellulose membrane (catalog M06584) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M06584) |
| Primary antibody | M06584 · 1:500 (catalog M06584) |
| Primary incubation | overnight at 4°C (catalog M06584) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M06584) |
| Secondary incubation | 1.5 hour at RT (catalog M06584) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M06584) |
| Detection | ECL (catalog M06584) |
Section 2
What Is the Expected PSMD14 Western Blot Band Size?
PSMD14 is predicted at 34.6 kDa and observed at ~35 kDa; the small difference has no established cause in the supplied features.
| Single band at ~35 kDa | Matches the empirical PSMD14 band near its 34.6 kDa predicted mass; confirm identity with a PSMD14 control |
| Slight mobility variation near 35 kDa | Phosphorylation at Ser150, Ser224, or Thr266 could affect migration, but no shift is established |
| Nearby doublet around 35 kDa | Different phosphorylation states are possible, but their resolution as separate bands is unproven |
| High molecular weight signal under native conditions | Could reflect PSMD14 within the proteasome regulatory particle; complex migration is not established for this blot |
💡Expected PSMD14 appearancePSMD14 has a predicted mass of 34.6 kDa and an empirical band at ~35 kDa in reducing whole-cell blots; verify band identity with a PSMD14 depletion or independent antibody control.
| Predicted PSMD14 mass | 34.6 kDa predicted; the observed band is ~35 kDa |
| Phosphoserine at Ser150 | Could alter mobility slightly; a visible shift is not established |
| Phosphoserine at Ser224 | Could alter mobility slightly; a visible shift is not established |
| Phosphothreonine at Thr266 | Could alter mobility slightly; a visible shift is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | Phosphorylation could affect mobility, but a large shift has no established feature-based explanation | Compare with the ~35 kDa reference band and verify identity by PSMD14 depletion |
| Band lower than expected | Possible degradation or an unrelated antibody-reactive protein; no cleavage feature is listed | Use fresh lysate with protease inhibitors and verify identity by PSMD14 depletion |
| Multiple bands | Different phosphorylation states are possible, but distinct bands are unproven | Compare phosphatase-treated and untreated lysates, then confirm candidate bands by PSMD14 depletion |
| Weak or no signal | PSMD14 detection or sample preparation may be inadequate | Check loading and transfer, then run a lysate known to show the ~35 kDa band |
| Fragments below expected size | Possible sample degradation; no precursor cleavage is listed | Prepare fresh lysate with protease inhibitors and check whether the fragments track with PSMD14 depletion |
Experiment evidence
Western blot images showing PSMD14 band
M06584 · PSMD14
M06584 · PSMD14
A06584-1 · PSMD14
A06584-1 · PSMD14
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PSMD14 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PSMD14 in Western blot, you can use adipose tissue, which HPA scores as medium expression.
Positive control: Adipose tissue (IHC candidate; verify WB)
Negative control: Fallopian tube (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: HPA reports medium expression in the positive tissue and no detection in fallopian tube, so verify the contrast in your samples.
HPA tissue expression evidence for PSMD14
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 |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Caudate | neuronal cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Fallopian tube | glandular cells | Not detected | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Not detected | Protein (IHC) | HPA → |
| Prostate | glandular cells | Not detected | Protein (IHC) | HPA → |
| Salivary gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PSMD14 Western Blot Tips
Deeper troubleshooting and optimisation questions for PSMD14, answered from its protein features.
How should PSMD14 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.
Are multiple PSMD14 isoforms expected?
Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. Do not assign additional bands to documented PSMD14 isoforms on this evidence.
Could phosphorylation explain a shifted PSMD14 band?
PTM · UniProt lists phosphoserine at positions 150 and 224 and phosphothreonine at position 266. These are UniProt coordinates; antibody or paper numbering may differ. Site presence alone does not establish a visible shift or explain a mass difference.
No glycosylation sites are listed, and the glycosylation count is zero. The supplied features therefore do not support glycosylation as an explanation for a higher band.
No glycosylation sites are listed, and the glycosylation count is zero. The supplied features therefore do not support glycosylation as an explanation for a higher band.
Does this guide establish induction of PSMD14?
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 PSMD14?
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 M06584 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 PSMD14 be quantified across samples?
Quantitation · Quantify the band near 35 kDa consistently across samples, using the same band boundaries and a validated loading normalization. PSMD14 is a component of the 19S proteasome regulatory particle, so a change in its band intensity alone does not establish a change in whole-proteasome activity.
How does the observed PSMD14 band compare with its predicted mass?
Interpretation · The observed band near 35 kDa is close to the predicted 34.6 kDa. This agreement supports the assignment, but apparent mass alone does not establish band identity.
Check whether the expected band near 35 kDa is present before assigning other bands. The record documents one isoform, three phosphorylation sites, and membership in the 19S regulatory particle; none of these features alone identifies an unexpected band.
Check whether the expected band near 35 kDa is present before assigning other bands. The record documents one isoform, three phosphorylation sites, and membership in the 19S regulatory particle; none of these features alone identifies an unexpected band.
Boster reagents
PSMD14 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-PSMD14 Rabbit Monoclonal Antibody
Cat # M06584
Real WB data
Anti-POH1/PSMD14 Antibody Picoband®
Cat # A06584-1
Two the supplier anti-PSMD14 antibodies have WB images. M06584 shows an approximately 35 kDa band in human cell and rat and mouse tissue lysates; A06584-1 shows an approximately 36 kDa band in human cell lysates. Neither image provides independent specificity validation.
Which to pick: For rat or mouse tissue lysates, M06584 has directly shown WB examples. For human cell lysates, both have images; use the reported starting conditions: M06584 at 1:500 or A06584-1 at 0.5 μg/mL.
Source: BosterBio PSMD14 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O00487.
- Human Protein Atlas. PSMD14 tissue expression.
- PMC10407523 — target-verified WB comparison
- PMC13281508 — target-verified WB comparison
- PMC12087398 — target-verified WB comparison