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- Table of Contents
VHL / von Hippel-Lindau disease tumor suppressor · Western blot design guide
Design a Western Blot for VHL
Real validated VHL Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-VHL 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
VHL Western Blot Experimental Design Guide
Expected bands, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~24.2 kDa | |
| Observed band | ~18 kDa | |
| Gel | 13% (catalog A00079-3) | |
| Positive control | Appendix (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 | — | |
| Caveat | Alternative initiation | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated VHL Western Blot Protocols
The A00079-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Jurkat, human Raji, human Hela (catalog A00079-3) |
| Gel % | 13% (catalog A00079-3) |
| Load | 30 ug; reducing conditions (catalog A00079-3) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00079-3) |
| Membrane | nitrocellulose membrane (catalog A00079-3) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00079-3) |
| Primary antibody | A00079-3 · 1:1000 (catalog A00079-3) |
| Primary incubation | overnight at 4°C (catalog A00079-3) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00079-3) |
| Secondary incubation | 1.5 hour at RT (catalog A00079-3) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00079-3) |
| Detection | ECL (catalog A00079-3) |
Section 2
What Is the Expected VHL Western Blot Band Size?
VHL is predicted at 24.2 kDa, but antibody QC observes ~18 kDa in reducing whole-cell lysates; the cause of the difference is not established.
| Band at ~18 kDa | Empirical VHL band in reducing whole-cell lysates; the difference from predicted mass is unexplained |
| Band near 24.2 kDa | Near the predicted VHL mass; confirm identity with band controls |
| Several discrete bands | Isoforms 1, 2, and 3 are documented, but distinct migration is unproven |
| Weak band in one subcellular fraction | Isoform 1 is reported in several cellular compartments |
💡Expected VHL appearanceVHL has a predicted mass of 24.2 kDa, while antibody QC shows a ~18 kDa band in reducing whole-cell lysates; the difference is unexplained, so confirm identity with appropriate controls.
| UniProt predicted mass | 24.2 kDa is the sequence-based reference, not the observed migration |
| Isoform 1 | Its individual mass and migration are not supplied |
| Isoform 2 | Its individual mass and migration are not supplied |
| Isoform 3 | Its individual mass and migration are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | VHL signal may be below detection in the tested lysate | Check transfer and compare with Jurkat, Raji, or HeLa whole-cell lysate |
| Band higher than expected | The higher band's identity is unestablished; VHL has three named isoforms | Compare with the ~18 kDa QC band and test identity with a second antibody or VHL knockdown |
| Band lower than expected | The observed ~18 kDa VHL band lies below the 24.2 kDa prediction for an unestablished reason | Compare with the reducing whole-cell QC pattern and verify with VHL knockdown |
| Multiple bands | Isoforms are documented, but their separate band positions are unknown | Test band identity with a second antibody or isoform-specific controls |
| Weak or no signal | Isoform 1 occurs in cytoplasm, membrane, endoplasmic reticulum, and nucleus | Compare the tested fraction with whole-cell lysate and check loading and transfer |
Experiment evidence
Western blot images showing VHL band
A00079-3 · VHL
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for VHL Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for VHL in Western blot, you can use appendix tissue, which HPA scores High.
Positive control: Appendix (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: HPA lists high and not-detected tissues, making tissue lysates practical positive and negative controls.
HPA tissue expression evidence for VHL
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 |
|---|---|---|---|---|
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cervix | squamous epithelial 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 → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Soft tissue | fibroblasts | Not detected | Protein (IHC) | HPA → |
| Vagina | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced VHL Western Blot Tips
Deeper troubleshooting and optimisation questions for VHL, answered from its protein features.
How should VHL 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 VHL isoforms affect the band pattern?
Isoforms · UniProt lists three isoforms. Relative to the canonical sequence, isoform 3 lacks residues 1–53 and isoform 2 lacks residues 114–154, using UniProt coordinates. Check whether the antibody epitope lies within either missing region before interpreting multiple bands or comparing isoform signals.
Can a listed modification explain a VHL band shift?
PTM · The supplied features list no modified residues or glycosylation sites. They therefore provide no basis for assigning a VHL band shift to a specific modification. Even an annotated modification would not, by itself, establish a visible shift.
Does this guide establish induction of VHL?
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.
What transfer method to use for VHL Western blot?
Transfer · Optimize transfer for the ~18 kDa band as well as the predicted 24.2 kDa protein. Use a membrane suited to retaining small proteins, and check the post-transfer gel and membrane to assess recovery. The supplied features do not establish a single best transfer condition.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A00079-3 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 VHL bands be quantified across samples?
Quantitation · Define which band or isoform the antibody measures before comparing samples, and quantify within the assay's linear range. Keep sample fraction and loading approach consistent: isoform 1 is reported in the cytoplasm, nucleus, endoplasmic reticulum, and at the cell membrane.
Why might VHL appear near 18 kDa instead of 24.2 kDa?
Interpretation · The supplied apparent band is ~18 kDa, while the predicted mass is 24.2 kDa. VHL has three isoforms, but these features alone cannot identify the ~18 kDa band or explain the mass difference. Confirm band identity with an isoform-aware antibody or another specific validation approach.
First compare their pattern with the three listed isoforms and the antibody's epitope. Isoforms 2 and 3 have different missing sequence regions in UniProt coordinates, but band position alone cannot establish identity. VHL is also a component of the VCB complex; the supplied features do not identify additional band masses.
First compare their pattern with the three listed isoforms and the antibody's epitope. Isoforms 2 and 3 have different missing sequence regions in UniProt coordinates, but band position alone cannot establish identity. VHL is also a component of the VCB complex; the supplied features do not identify additional band masses.
Boster reagents
VHL 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-VHL Antibody Picoband®
Cat # A00079-3
A00079-3 is a rabbit polyclonal anti-VHL antibody listed for human, mouse, and rat. Its Western blot image shows human Jurkat, Raji, and HeLa lysates, with a band near 18 kDa versus an expected 20 kDa. The supplied image does not document mouse or rat samples.
Which to pick: A00079-3 is the only listed option. Its Western blot example uses human cell lysates at 1:1000 primary antibody dilution. Mouse and rat are listed as reactive, but no Western blot image for those samples is supplied.
Source: BosterBio VHL gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P40337.
- Human Protein Atlas. VHL tissue expression.
- PMC12003641 — target-verified WB comparison
- PMC7368819 — target-verified WB comparison