This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
VPS35 / Vacuolar protein sorting-associated protein 35 · Western blot design guide
Design a Western Blot for VPS35
Real validated VPS35 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-VPS35 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
VPS35 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 | ~91.7 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Skeletal muscle (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated VPS35 Western Blot Protocols
The M01644 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | (1) HeLa cell lysate; (2) Mouse kidney lysate (catalog M01644) |
| Gel % | 8–10% (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 | M01644; 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 VPS35 Western Blot Band Size?
Full-length VPS35 is predicted at 91.7 kDa; phosphorylation and localization are documented, but altered migration has not been demonstrated.
| Single band near 91.7 kDa | consistent with full-length VPS35, pending identity controls |
| Band in whole-cell lysate | consistent with cytoplasmic VPS35 |
| Band in an endosome-enriched fraction | consistent with VPS35 endosomal localization |
| Band in a membrane fraction | consistent with peripheral membrane-associated VPS35 |
💡Expected VPS35 appearanceVPS35 has a predicted mass of 91.7 kDa, but no empirical band size is supplied; confirm any candidate band with ordinary identity controls.
| Predicted molecular mass | Places full-length VPS35 near 91.7 kDa |
| Ser7 phosphorylation | No apparent size change is established |
| Ser783 phosphorylation | No apparent size change is established |
| Tyr791 phosphorylation | No apparent size change is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | VPS35 signal may be lost during preparation of an endosome-associated fraction | Check both whole-cell lysate and retained membrane fractions |
| Band higher than expected | The cause of migration above 91.7 kDa is not established | Compare with a molecular-weight marker and verify identity by VPS35 depletion |
| Band lower than expected | A smaller band could reflect degradation or nonspecific binding | Use fresh lysate and check whether VPS35 depletion removes the band |
| Multiple bands | Additional bands have no established isoform explanation | Use VPS35 depletion to identify the specific band |
| Weak or no signal | Sample preparation may recover little endosome-associated VPS35 | Check lysate loading and compare whole-cell with membrane fractions |
Experiment evidence
Western blot images showing VPS35 band
M01644 · VPS35
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for VPS35 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for VPS35 in Western blot, you can use appendix lysate, which HPA scores High.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Skeletal muscle (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: VPS35 is intracellular, and HPA identifies skeletal muscle as a feasible negative tissue control.
HPA tissue expression evidence for VPS35
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 → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular cells | High | Protein (IHC) | HPA → |
| Endometrium | glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Skeletal muscle | myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Not detected | Protein (IHC) | HPA → |
| Liver | hepatocytes | Low | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced VPS35 Western Blot Tips
Deeper troubleshooting and optimisation questions for VPS35, answered from its protein features.
How should VPS35 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 VPS35 isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. Do not assign additional bands to isoforms from this evidence alone.
Which VPS35 phosphorylation sites matter when interpreting band shifts?
PTM · UniProt lists phosphoserine at positions 7 and 783 and phosphotyrosine at position 791. These are UniProt coordinates; paper or antibody numbering may differ. Their presence does not establish a visible band shift.
Does this guide establish induction of VPS35?
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 VPS35 Western blot?
Transfer · VPS35 is predicted to be 91.7 kDa. Validate that your chosen transfer conditions recover protein near this mass on the membrane. The supplied features do not identify a preferred transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M01644 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 VPS35 abundance be quantified across samples?
Quantitation · VPS35 is found in the cytoplasm and on endosomal membranes. Compare equally prepared samples or matched cellular fractions, and use a loading control appropriate to the material measured; fraction differences can affect the VPS35 signal.
Should VPS35 migrate exactly at its predicted mass?
Interpretation · VPS35 has a predicted mass of 91.7 kDa, but no observed band position is supplied. Compare your band with a molecular weight marker; the listed features alone cannot explain a difference between apparent and predicted mass.
Check their apparent masses against the predicted 91.7 kDa mass and confirm identity independently. The single listed isoform and three phosphorylation sites do not, by themselves, identify the cause of additional bands.
Check their apparent masses against the predicted 91.7 kDa mass and confirm identity independently. The single listed isoform and three phosphorylation sites do not, by themselves, identify the cause of additional bands.
Boster reagents
VPS35 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-VPS35 Monoclonal Antibody
Cat # M01644
The catalog reports anti-VPS35 monoclonal antibody M01644 for Western blotting, with reported Human, Mouse, and Rat reactivity. Its WB image shows HeLa cell and mouse kidney lysates; the supplied evidence does not show a rat sample.
Which to pick: M01644 is the only listed option. Its WB image includes HeLa and mouse kidney lysates, making those the directly shown sample contexts; Rat is listed in reactivity but is not shown in the supplied image description.
Source: BosterBio VPS35 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q96QK1.
- Human Protein Atlas. VPS35 tissue expression.
- PMC5477581 — target-verified WB comparison
- PMC6949432 — target-verified WB comparison
- PMC13337223 — target-verified WB comparison