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CBLB / E3 ubiquitin-protein ligase CBL-B · Western blot design guide
Design a Western Blot for CBLB
Source-linked CBLB Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CBLB 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
CBLB 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 | ~109.5 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Adrenal gland (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 Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked CBLB Western Blot Protocol Options
The A00735 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | extracts of various cell lines, (catalog A00735) |
| 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 | Blocking buffer: 3% nonfat dry milk in TBST (catalog A00735) |
| Primary antibody | A00735 · 1:1000 (catalog A00735) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Goat Anti-Rabbit IgG, 1:10000 (catalog A00735) |
| 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 CBLB Western Blot Band Size?
CBLB is predicted at 109.5 kDa; documented isoforms and phosphorylation could affect the pattern, but their effects on migration are unverified.
| Band near 109.5 kDa | Consistent with the predicted size of CBLB; confirm its identity with controls. |
| Band below 109.5 kDa | Could represent Truncated 1 or Truncated 2; identity requires verification. |
| Several bands at different positions | May include CBLB isoforms, though distinct migration has not been demonstrated. |
| Close doublet near 109.5 kDa | Could reflect documented phosphorylation; a visible shift has not been established. |
💡Expected CBLB appearanceUniProt predicts 109.5 kDa for CBLB; no empirical band size is supplied, and any additional bands require identity controls because isoform separation and phosphorylation-dependent migration are unverified.
| UniProt predicted mass | Provides a 109.5 kDa reference, not a measured band position. |
| Long isoform | Its apparent band size is not supplied. |
| Truncated 1 isoform | Its truncated sequence may yield a lower band; migration is not documented. |
| Truncated 2 isoform | Its truncated sequence may yield a lower band; migration is not documented. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CBLB may be below detection in the sampled cytoplasmic lysate. | Check a positive-control lysate and antibody performance. |
| Band higher than expected | Documented phosphorylation could affect migration, but no shift is established. | Compare phosphatase-treated and untreated samples and confirm band identity. |
| Band lower than expected | Truncated 1 or Truncated 2 may contribute. | Check isoform recognition and confirm the band with an independent antibody. |
| Multiple bands | Long, Truncated 1 and Truncated 2 isoforms are documented; their separation is unknown. | Compare isoform-specific controls and verify band identity. |
| Weak or no signal | CBLB may be scarce or poorly detected in the tested sample. | Check sample loading and a positive-control lysate. |
Experiment evidence
Western blot images showing CBLB band
A00735 · CBLB
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CBLB Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CBLB in Western blot, you can use adrenal gland tissue.
Positive control: Adrenal gland (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: Cytoplasmic CBLB should be detectable in tissue lysate, and HPA lists adipose tissue as not detected.
HPA tissue expression evidence for CBLB
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 | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | High | Protein (IHC) | HPA → |
| Colon | glandular 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 → |
| Epididymis | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Fallopian tube | glandular cells | Low | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced CBLB Western Blot Tips
Deeper troubleshooting and optimisation questions for CBLB, answered from its protein features.
How should CBLB 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 CBLB isoforms produce multiple bands?
Isoforms · Three isoforms are listed: Long, Truncated 1 and Truncated 2. Relative to the canonical UniProt sequence, Truncated 1 lacks residues 811–982; Truncated 2 changes residues 767–770 from DVFD to TYRI and lacks 771–982. Their distinct lengths could yield different bands, but no observed positions are supplied.
Choose the band or bands that match the isoform being measured and apply the same choice across samples. The two truncated isoforms lack different portions of the canonical C terminus, so check whether the antibody target region is retained before combining signals.
Choose the band or bands that match the isoform being measured and apply the same choice across samples. The two truncated isoforms lack different portions of the canonical C terminus, so check whether the antibody target region is retained before combining signals.
Which phosphorylation sites matter when interpreting CBLB bands?
PTM · UniProt lists phosphoserines at 282, 476, 480, 484, 521, 525, 529 and 634, and phosphotyrosines at 363, 665, 709 and 889. Ser282 is annotated as phosphorylated by PKC/PRKCQ. These are UniProt canonical-sequence coordinates; antibody or paper numbering may differ. Site annotations alone do not establish a visible shift.
CBLB has 12 annotated phosphorylation sites, including Ser282 modified by PKC/PRKCQ. A modification could affect migration, but the annotations do not demonstrate a visible shift or explain a difference from the calculated 109.5 kDa mass. Compare appropriately treated samples before assigning a shifted band to phosphorylation.
CBLB has 12 annotated phosphorylation sites, including Ser282 modified by PKC/PRKCQ. A modification could affect migration, but the annotations do not demonstrate a visible shift or explain a difference from the calculated 109.5 kDa mass. Compare appropriately treated samples before assigning a shifted band to phosphorylation.
Does EGF stimulation change CBLB localization?
Induction · CBLB is annotated in the cytoplasm and associates with endocytic vesicles upon EGF stimulation. That supports comparing stimulated and unstimulated samples when studying localization; it does not establish a change in total CBLB abundance or phosphorylation.
How should transfer be checked for CBLB?
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 A00735 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 CBLB 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.
Where should full-length CBLB migrate on a Western blot?
Interpretation · The predicted mass of the 982-residue Long isoform is 109.5 kDa. No observed band position is supplied, so use this as a reference rather than an expected apparent mass.
Consider the annotated truncated isoforms: Truncated 1 lacks canonical residues 811–982, while Truncated 2 lacks 771–982 and changes 767–770. Check whether the antibody recognizes a region shared with those isoforms. Band position alone cannot identify an isoform.
Consider the annotated truncated isoforms: Truncated 1 lacks canonical residues 811–982, while Truncated 2 lacks 771–982 and changes 767–770. Check whether the antibody recognizes a region shared with those isoforms. Band position alone cannot identify an isoform.
Boster reagents
CBLB 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-CBLB Antibody
Cat # A00735
A00735 is an anti-CBLB antibody listed for human and mouse reactivity. Its WB image caption reports extracts from various cell lines, 25 µg per lane, and a 1:1000 antibody dilution; it does not name the cell lines or their species.
Which to pick: A00735 is the only listed CBLB antibody. It has a WB image with reported loading and dilution conditions; confirm suitability for your sample, since the caption does not identify the tested cell lines.
Source: BosterBio CBLB gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q13191.
- Human Protein Atlas. CBLB tissue expression.
- PMC9655234 — target-verified WB comparison
- PMC7986888 — target-verified WB comparison
- PMC13231350 — target-verified WB comparison