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USP21 / Ubiquitin carboxyl-terminal hydrolase 21 · Western blot design guide
Design a Western Blot for USP21
Source-linked USP21 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-USP21 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
USP21 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 | ~62.7 kDa | |
| Observed band | ~63 kDa | |
| Gel | 5–20% (catalog A06639-1) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Bone marrow (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | — | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked USP21 Western Blot Protocol Options
The A06639-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human HELA, human HEPG2, human PANC-1, human HEK293, rat kidney, mouse kidney, mouse RAW264.7 (catalog A06639-1) |
| Gel % | 5–20% (catalog A06639-1) |
| Load | 50ug; reducing conditions (catalog A06639-1) |
| Transfer | a Nitrocellulose membrane at 150mA for 50-90 minutes (catalog A06639-1) |
| Membrane | Nitrocellulose membrane (catalog A06639-1) |
| Blocking | 5% Non-fat Milk/ TBS for 1.5 hour at RT (catalog A06639-1) |
| Primary antibody | A06639-1 · 0.5 μg/mL (catalog A06639-1) |
| Primary incubation | overnight at 4°C (catalog A06639-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A06639-1) |
| Secondary incubation | 1.5 hour at RT (catalog A06639-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A06639-1) |
| Detection | ECL (catalog A06639-1) |
Section 2
What Is the Expected USP21 Western Blot Band Size?
USP21 is predicted at 62.7 kDa and observed near 63 kDa; the small difference has no established cause in the supplied evidence.
| Band near 63 kDa | Matches the reported USP21 band and its 62.7 kDa predicted mass; confirm identity with controls |
| Several bands at different positions | Could include USP21 isoforms 1, 2, and 3; their migration has not been established |
| Band in a cytoplasmic fraction | Consistent with reported cytoplasmic localization |
| Band in a nuclear fraction | Consistent with reported nuclear localization |
💡Expected USP21 appearanceUSP21 has a predicted mass of 62.7 kDa and an observed band near 63 kDa; confirm band identity with ordinary antibody controls, since isoform migration is not established.
| UniProt predicted mass | Predicts 62.7 kDa, close to the observed band near 63 kDa |
| Isoform 1 | Its individual mass and migration are not supplied |
| Isoform 2 | Alternative splicing may change its size, but its individual migration is not supplied |
| Isoform 3 | Alternative splicing may change its size, but its individual migration is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | USP21 signal may be below detection in the sampled lysate | Check loading and antibody performance with a positive control; examine cytoplasmic and nuclear fractions |
| Band higher than expected | The higher band's identity is unestablished by the supplied features | Compare with the 63 kDa reference and check specificity with an independent antibody or USP21 depletion |
| Band lower than expected | An alternative isoform is possible, but its migration is unknown | Check specificity with USP21 depletion or an isoform-specific control |
| Multiple bands | USP21 has isoforms 1, 2, and 3, but distinct bands are not established | Test band identity with USP21 depletion and isoform-specific controls |
| Weak or no signal | USP21 abundance in the sampled cytoplasmic or nuclear fraction may be low | Check loading, fraction recovery, and antibody performance with a positive control |
Experiment evidence
Western blot images showing USP21 band
A06639-1 · USP21
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for USP21 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for USP21 in Western blot, you can use adrenal gland lysate.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Bone marrow (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: HPA rates the positive tissue only Medium, so the signal may be modest.
HPA tissue expression evidence for USP21
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Medium | Protein (IHC) | HPA → |
| Endometrium | glandular cells | Medium | Protein (IHC) | HPA → |
| Gallbladder | glandular cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial 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 → |
Section 3
Advanced USP21 Western Blot Tips
Deeper troubleshooting and optimisation questions for USP21, answered from its protein features.
How should USP21 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 isoform 2 produce a smaller USP21 band?
Isoforms · Yes. Relative to the canonical UniProt sequence, isoform 2 replaces residues 372–382 (DLFVGQLKSCL with GMEWGKAMREN) and lacks residues 383–565. It could therefore yield a smaller band if expressed and recognized by the antibody. The features do not establish its abundance or apparent mass.
Isoform 3 lacks canonical UniProt residues 498–511. A band near the canonical band could be consistent with this isoform, but the deletion alone does not establish a resolvable shift. Check whether the antibody’s epitope includes residues 498–511.
Decide which bands the measurement represents and apply the same band boundaries across samples. Isoform 2 has a large deletion, while isoform 3 lacks canonical residues 498–511; antibody recognition may differ by epitope. Do not combine distinct bands as total USP21 without establishing that they represent USP21 isoforms.
Isoform 3 lacks canonical UniProt residues 498–511. A band near the canonical band could be consistent with this isoform, but the deletion alone does not establish a resolvable shift. Check whether the antibody’s epitope includes residues 498–511.
Decide which bands the measurement represents and apply the same band boundaries across samples. Isoform 2 has a large deletion, while isoform 3 lacks canonical residues 498–511; antibody recognition may differ by epitope. Do not combine distinct bands as total USP21 without establishing that they represent USP21 isoforms.
Can annotated modifications explain an unexpected USP21 band shift?
PTM · No modified residues or glycosylation sites are listed in the supplied features. These annotations provide no specific modification-based explanation for a shift. Compare candidate bands with the three annotated isoform sequences before assigning an unexpected band.
Does this guide establish induction of USP21?
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 USP21?
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 A06639-1 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 should USP21 be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
How should the ~63 kDa band compare with USP21’s predicted mass?
Interpretation · The observed ~63 kDa band closely matches the 62.7 kDa predicted mass for canonical USP21. Apparent mass alone does not establish band identity; check whether the antibody recognizes the canonical sequence and relevant isoforms.
USP21 is annotated in both cytoplasm and nucleus. Use comparable sample preparation across lanes, and interpret changes in a cellular fraction with that dual localization in mind. A difference in one fraction alone does not establish a change in total USP21.
USP21 is annotated in both cytoplasm and nucleus. Use comparable sample preparation across lanes, and interpret changes in a cellular fraction with that dual localization in mind. A difference in one fraction alone does not establish a change in total USP21.
Boster reagents
USP21 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-USP21 Antibody Picoband®
Cat # A06639-1
The catalog reports one anti-USP21 antibody, A06639-1, with a Western blot image showing an approximately 63 kDa band in human cell, rat kidney, mouse kidney, and mouse cell lysates. The supplied evidence includes no independent publication validation.
Which to pick: A06639-1 is the only listed option. It is labeled reactive with human, mouse, and rat USP21; its WB image includes examples from all three species. Match your sample to those tested contexts where possible.
Source: BosterBio USP21 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9UK80.
- Human Protein Atlas. USP21 tissue expression.
- PMC10989364 — target-verified WB comparison
- PMC5173112 — target-verified WB comparison
- PMC11895906 — target-verified WB comparison