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- Table of Contents
USP28 / Ubiquitin carboxyl-terminal hydrolase 28 · Western blot design guide
Design a Western Blot for USP28
Source-linked USP28 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-USP28 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
USP28 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 | ~122.5 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Bronchus (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Ubl conjugation | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked USP28 Western Blot Protocol Options
The M05040 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | A431 cell lysate (catalog M05040) |
| Gel % | 8–10% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M05040; 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 USP28 Western Blot Band Size?
USP28 is predicted at 122.5 kDa; isoforms and phosphorylation are annotated, but their effects on visible migration have not been demonstrated.
| Band near 122.5 kDa | Consistent with predicted USP28 size; confirm its identity with an appropriate control. |
| Several bands at different positions | Could reflect USP28 isoforms 1, 2, and 3; their migration has not been established. |
| Close doublet near 122.5 kDa | Could reflect annotated phosphorylation, but a visible shift has not been demonstrated. |
| Weak band in a cytoplasmic fraction | USP28 is annotated in the nucleoplasm. |
💡Expected USP28 appearanceUSP28 has a predicted mass of 122.5 kDa, but no empirical band size is supplied; confirm any candidate band with an appropriate identity control.
| Predicted USP28 mass | Places the reference size at 122.5 kDa, without establishing apparent migration. |
| Isoform 1 | Its individual mass and migration are not supplied. |
| Isoform 2 | Could migrate differently from other isoforms; its mass is not supplied. |
| Isoform 3 | Could migrate differently from other isoforms; its mass is not supplied. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nucleoplasmic USP28 may be poorly recovered during extraction. | Check nuclear protein recovery and antibody performance with a positive control. |
| Band higher than expected | The supplied features do not establish a larger migrating form. | Check band identity with an independent antibody or USP28 depletion. |
| Band lower than expected | An isoform is possible, but isoform masses are not supplied. | Check band identity with USP28 depletion or an isoform-aware reagent. |
| Multiple bands | USP28 has three annotated splice isoforms; their separation is unknown. | Compare bands after USP28 depletion and, if available, with isoform-specific reagents. |
| Weak or no signal | Nucleoplasmic USP28 may be underrepresented in the sample. | Assess nuclear extraction and include a positive-control lysate. |
Experiment evidence
Western blot images showing USP28 band
M05040 · USP28
M05040-1 · USP28
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for USP28 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for USP28 in Western blot, you can use bronchus tissue, which HPA reports as highly expressing USP28.
Positive control: Bronchus (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: USP28 is nucleoplasmic, so a nuclear-enriched lysate may improve detection.
HPA tissue expression evidence for USP28
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 |
|---|---|---|---|---|
| Bronchus | basal cells | High | Protein (IHC) | HPA → |
| Cervix | glandular cells | High | Protein (IHC) | HPA → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | High | Protein (IHC) | HPA → |
| Nasopharynx | basal cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | fibroblasts | Not detected | Protein (IHC) | HPA → |
| Vagina | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced USP28 Western Blot Tips
Deeper troubleshooting and optimisation questions for USP28, answered from its protein features.
How should USP28 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.
How could USP28 isoforms affect band patterns?
Isoforms · UniProt lists isoforms 1, 2, and 3. Isoform 2 lacks residues 769–800; isoform 3 changes residue 583 from P to E and lacks residues 584–1077. An antibody whose binding region falls in a deleted segment may miss that isoform. These features identify possible band differences but do not establish which isoforms are present in a sample.
Isoform 3 lacks canonical residues 584–1077, so it lacks the annotated sites at 714 and 1048. Its sites at 67, 375, and 550 lie before the deletion. Isoform 2 deletes canonical residues 769–800, outside the five listed sites. Use canonical UniProt coordinates when matching these annotations to isoforms; positions after a deletion may be numbered differently in an isoform sequence.
Isoform 3 lacks canonical residues 584–1077, so it lacks the annotated sites at 714 and 1048. Its sites at 67, 375, and 550 lie before the deletion. Isoform 2 deletes canonical residues 769–800, outside the five listed sites. Use canonical UniProt coordinates when matching these annotations to isoforms; positions after a deletion may be numbered differently in an isoform sequence.
Which USP28 phosphorylation sites are annotated?
PTM · The supplied UniProt features list phosphoserine at positions 67, 375, 550, and 714, and phosphothreonine at 1048. These are canonical UniProt coordinates; check the numbering convention used for any phosphosite antibody before comparing positions.
Does this guide establish induction of USP28?
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 USP28?
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 M05040 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 USP28 be quantified when multiple bands appear?
Quantitation · Decide which band or bands you will quantify based on the isoforms your antibody can recognize. Isoforms 2 and 3 contain deletions, so pooling all bands could obscure isoform differences. Report the quantified band positions and use the same selection across samples. The supplied features provide no observed USP28 band size to use as a definitive assignment.
Why might USP28 bands differ from 122.5 kDa?
Interpretation · 122.5 kDa is the predicted mass for canonical USP28. Isoform 2 lacks residues 769–800, and isoform 3 has a P-to-E substitution at residue 583 and lacks residues 584–1077. These sequence differences could affect band position. The annotated phosphorylation sites alone do not establish a visible shift, and no observed band size is supplied.
USP28 has a DNA damage annotation, and the supplied interaction record says it dissociates from FBXW7 following DNA damage. That supports considering DNA damage when comparing conditions, but it does not establish increased USP28 abundance, phosphorylation, or a band shift. Measure treated and control samples before drawing those conclusions.
Isoform 2 lacks 32 residues at canonical positions 769–800, while isoform 3 lacks residues 584–1077 and should be substantially shorter in sequence. These are candidates for a lower band, but band position alone cannot identify an isoform. Check whether the antibody binding region is retained in the candidate isoform.
USP28 has a DNA damage annotation, and the supplied interaction record says it dissociates from FBXW7 following DNA damage. That supports considering DNA damage when comparing conditions, but it does not establish increased USP28 abundance, phosphorylation, or a band shift. Measure treated and control samples before drawing those conclusions.
Isoform 2 lacks 32 residues at canonical positions 769–800, while isoform 3 lacks residues 584–1077 and should be substantially shorter in sequence. These are candidates for a lower band, but band position alone cannot identify an isoform. Check whether the antibody binding region is retained in the candidate isoform.
Boster reagents
USP28 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-USP28 Monoclonal Antibody
Cat # M05040
Real WB data
Anti-USP28 Rabbit Monoclonal Antibody
Cat # M05040-1
The catalog reports two anti-USP28 antibodies with Western blot images: M05040 in A431 lysate and M05040-1 in SW480 lysate. Both images use human cell lines; the supplied evidence does not show Western blots in mouse or rat samples.
Which to pick: For human samples, choose based on the tested lysate closest to your experiment: M05040 has an A431 blot, while M05040-1 has a SW480 blot. M05040-1 also lists mouse and rat reactivity, but no Western blot images for those species are supplied.
Source: BosterBio USP28 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q96RU2.
- Human Protein Atlas. USP28 tissue expression.
- PMC8901929 — target-verified WB comparison
- PMC8478945 — target-verified WB comparison
- PMC6317191 — target-verified WB comparison