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- Table of Contents
PSMD4 / 26S proteasome non-ATPase regulatory subunit 4 · Western blot design guide
Design a Western Blot for PSMD4
Source-linked PSMD4 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PSMD4 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
PSMD4 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 | ~40.7 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Caudate (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 + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked PSMD4 Western Blot Protocol Options
The M03544-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | K562 cell lysate (catalog M03544-1) |
| 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 | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M03544-1; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected PSMD4 Western Blot Band Size?
PSMD4 is predicted at 40.7 kDa; splice isoforms could affect migration, but no empirical band size or distinct isoform pattern is established.
| Band near 40.7 kDa | Consistent with the predicted PSMD4 mass; confirm identity with controls |
| Single band near 40.7 kDa | Rpn10A and Rpn10E need not resolve as separate bands |
| Several bands near the expected region | Rpn10A and Rpn10E are possible contributors, but their migration is unknown |
| Band far from 40.7 kDa | Its identity cannot be established from the supplied features alone |
💡Expected PSMD4 appearancePSMD4 has a predicted mass of 40.7 kDa, but no empirical band size is supplied; use antibody and PSMD4 depletion controls to identify any band seen.
| UniProt predicted mass | 40.7 kDa provides a reference for the protein sequence |
| Rpn10A splice isoform | May differ in size from Rpn10E; its mass and migration are unspecified |
| Rpn10E splice isoform | May differ in size from Rpn10A; its mass and migration are unspecified |
| Alternative splicing of Rpn10A and Rpn10E | Could produce different band positions, but distinct bands are not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | PSMD4 abundance or detection may be insufficient | Check loading and antibody performance with a positive lysate |
| Band higher than expected | The supplied features do not establish its identity or shift | Check specificity with PSMD4 depletion and a molecular weight marker |
| Band lower than expected | An isoform or unrelated band is possible; isoform masses are unknown | Compare with PSMD4 depletion and an antibody to another epitope |
| Multiple bands | Rpn10A and Rpn10E are annotated, but their separation is unverified | Use isoform-specific evidence or PSMD4 depletion to assign bands |
| Weak or no signal | Low target abundance or inadequate antibody detection | Check loading, transfer, and a positive lysate |
| Fragments below expected size | Sample degradation is possible; no cleavage product is specified | Prepare fresh lysate with protease inhibitors and compare samples |
Experiment evidence
Western blot images showing PSMD4 band
M03544-1 · PSMD4
A03544-1 · PSMD4
A03544-1 · PSMD4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PSMD4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PSMD4 in Western blot, you can use caudate tissue, which HPA rates high for PSMD4.
Positive control: Caudate (IHC candidate; verify WB)
Negative control: Heart muscle (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside.
⚠️Feasibility: HPA lists heart muscle as not detected, but confirm the negative result in your lysate.
HPA tissue expression evidence for PSMD4
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 |
|---|---|---|---|---|
| Caudate | neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | GLUC cells - nucleus | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | High | Protein (IHC) | HPA → |
| Colon | endothelial cells | High | Protein (IHC) | HPA → |
| Hippocampus | neuronal 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 → |
| Nasopharynx | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PSMD4 Western Blot Tips
Deeper troubleshooting and optimisation questions for PSMD4, answered from its protein features.
How should PSMD4 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 PSMD4 isoforms produce different bands?
Isoforms · Two isoforms are listed: Rpn10A and Rpn10E. In Rpn10E, residues 255–268 are replaced and residues 269–377 are missing. A shorter band is plausible, but the features do not establish where either isoform runs on a blot.
Check the antibody epitope against the isoform sequences. Canonical residues 1–254 are shared; residues 255–268 differ in Rpn10E, and residues 269–377 are absent. An antibody targeting the absent region would not detect Rpn10E.
Check the antibody epitope against the isoform sequences. Canonical residues 1–254 are shared; residues 255–268 differ in Rpn10E, and residues 269–377 are absent. An antibody targeting the absent region would not detect Rpn10E.
Which PSMD4 phosphorylation sites matter when interpreting bands?
PTM · The supplied UniProt coordinates list phosphothreonine at 250 and 253 and phosphoserine at 256, 266, 358, and 361. These are canonical sequence coordinates; antibody or paper numbering may differ. Their presence alone does not demonstrate a visible band shift.
Canonical residues 250 and 253 precede Rpn10E’s altered region. Residues 256 and 266 lie within its replacement sequence, while 358 and 361 lie in the missing region. Do not assign the canonical phosphorylation sites in those regions to Rpn10E without isoform-specific evidence.
Canonical residues 250 and 253 precede Rpn10E’s altered region. Residues 256 and 266 lie within its replacement sequence, while 358 and 361 lie in the missing region. Do not assign the canonical phosphorylation sites in those regions to Rpn10E without isoform-specific evidence.
Does this guide establish induction of PSMD4?
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 PSMD4?
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 M03544-1 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 can PSMD4 bands be quantified across samples?
Quantitation · Use a consistent antibody and identify which isoform bands it detects before comparing signals. The Rpn10E sequence differs at 255–268 and lacks 269–377, so an epitope in those regions can bias an apparent total-PSMD4 measurement. The supplied features provide no basis for treating phosphorylation as a measured change in abundance.
Should PSMD4 run at its predicted 40.7 kDa?
Interpretation · The 377-residue canonical sequence has a predicted mass of 40.7 kDa. No observed band position is supplied, so the features cannot establish its apparent Western blot mass or explain a difference.
Consider Rpn10E for a shorter band, then check whether the antibody epitope is present in that isoform. PSMD4 is a phosphoprotein and has Ubl-conjugation and isopeptide-bond keywords, but no observed band positions or conjugation sites are supplied. Those annotations alone cannot identify an unexpected band.
Consider Rpn10E for a shorter band, then check whether the antibody epitope is present in that isoform. PSMD4 is a phosphoprotein and has Ubl-conjugation and isopeptide-bond keywords, but no observed band positions or conjugation sites are supplied. Those annotations alone cannot identify an unexpected band.
Boster reagents
PSMD4 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-PSMD4 Rabbit Monoclonal Antibody
Cat # M03544-1
Real WB data
Anti-PSMD4 Antibody Picoband®
Cat # A03544-1
Two the supplier anti-PSMD4 antibodies list human, mouse, and rat reactivity and have Western blot images. M03544-1 shows K562 lysate; A03544-1 shows human, rat, and mouse cell lysates. A03544-1 reports a band near 55 kDa versus an expected 41 kDa.
Which to pick: Choose A03544-1 for the documented human, rat, and mouse lysate examples and detailed blot conditions; account for its reported 55 kDa band. M03544-1 has a K562 lysate blot and is a rabbit monoclonal option.
Source: BosterBio PSMD4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P55036.
- Human Protein Atlas. PSMD4 tissue expression.
- PMC11900748 — target-verified WB comparison
- PMC7585163 — target-verified WB comparison
- PMC8797679 — target-verified WB comparison