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VAPA / Vesicle-associated membrane protein-associated protein A · Western blot design guide
Design a Western Blot for VAPA
Real validated VAPA Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-VAPA 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
VAPA 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 | ~27.9 kDa | |
| Observed band | ~33 kDa | |
| Gel | 5–20% (catalog A05329-1) | |
| 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 + Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated VAPA Western Blot Protocols
The A05329-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human K562, human 293T (catalog A05329-1) |
| Gel % | 5–20% (catalog A05329-1) |
| Load | 30 ug; reducing conditions (catalog A05329-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A05329-1) |
| Membrane | nitrocellulose membrane (catalog A05329-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A05329-1) |
| Primary antibody | A05329-1 · 0.5 μg/mL (catalog A05329-1) |
| Primary incubation | overnight at 4°C (catalog A05329-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A05329-1) |
| Secondary incubation | 1.5 hour at RT (catalog A05329-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A05329-1) |
| Detection | ECL (catalog A05329-1) |
Section 2
What Is the Expected VAPA Western Blot Band Size?
VAPA is predicted at 27.9 kDa but observed near 33 kDa on reducing blots; the cause of that difference is not established.
| Band near 33 kDa | Empirical VAPA band, compared with a predicted mass of 27.9 kDa |
| Band near twice the monomer position under nonreducing conditions | Possible Cys60-linked VAPA homodimer |
| Additional discrete band | Could reflect isoform 1 or 2, if they migrate differently |
| Weak or absent band in a soluble fraction | VAPA is an endoplasmic reticulum membrane protein |
💡Expected VAPA appearanceVAPA has a predicted mass of 27.9 kDa, while reducing Western blots show a band near 33 kDa; use an antibody specificity control to confirm band identity.
| Predicted VAPA mass | 27.9 kDa by sequence; the empirical reducing-blot band is near 33 kDa |
| Cys60 inter-chain disulfide | Can retain a species near twice the monomer position without complete reduction |
| Disulfide-linked homodimer | Can migrate above the monomer under nonreducing conditions |
| Splice isoforms 1 and 2 | May differ in mass, but their individual masses and separation on a blot are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated VAPA may be poorly recovered during extraction | Check membrane solubilization and include a membrane fraction |
| Band higher than expected | Cys60-linked homodimer may persist after incomplete reduction | Compare reducing and nonreducing lanes |
| Band lower than expected | A smaller band could reflect degradation or an isoform; its identity is unestablished | Use fresh lysate and an antibody specificity control |
| Multiple bands | VAPA has two splice isoforms and can form a disulfide-linked homodimer | Compare reducing and nonreducing lanes and check band identity |
| Weak or no signal | Membrane-associated VAPA may be underrepresented in the prepared fraction | Verify extraction and loading with a membrane protein control |
Experiment evidence
Western blot images showing VAPA band
A05329-1 · VAPA
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for VAPA Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for VAPA in Western blot, you can use adrenal gland lysate, which HPA rates High.
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: As an ER membrane protein, VAPA may require effective membrane solubilization for a reliable signal.
HPA tissue expression evidence for VAPA
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 → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal 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 → |
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Low | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Low | Protein (IHC) | HPA → |
| Caudate | glial cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced VAPA Western Blot Tips
Deeper troubleshooting and optimisation questions for VAPA, answered from its protein features.
How should VAPA 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 VAPA isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. At UniProt position 139, isoform 2 replaces L with LGITPPGNAPTVTSMSSINNTVATPASYHTKDDPRGLSVLKQEKQK. Compare antibody recognition of both sequences before assigning separate bands to isoforms; their listed sequences do not establish where each will migrate.
Which VAPA phosphorylation sites matter when interpreting bands?
PTM · UniProt lists phosphoserine at positions 166, 214, 216, and 219, and phosphothreonine at 170. These are UniProt coordinates; paper or antibody numbering may differ. If testing phosphorylation, check which site an antibody recognizes. The listed sites alone do not demonstrate a visible band shift.
UniProt lists N-acetylalanine at position 2 and N6-acetyllysine at position 125. These positions use UniProt numbering. Check an acetylation-specific antibody's stated target before interpreting its signal; these features alone do not predict a distinct band.
UniProt lists N-acetylalanine at position 2 and N6-acetyllysine at position 125. These positions use UniProt numbering. Check an acetylation-specific antibody's stated target before interpreting its signal; these features alone do not predict a distinct band.
Does this guide establish induction of VAPA?
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 VAPA Western blot?
Transfer · VAPA is a 249-residue, single-pass ER membrane protein; the supplied blots show an apparent band near 33 kDa. Check transfer efficiency around that position when optimizing your method. The supplied features do not specify an optimal transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A05329-1 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 VAPA bands be quantified?
Quantitation · Quantify the same verified band across samples, using the observed ~33 kDa position as a starting point. Because UniProt lists two isoforms, establish whether the antibody detects one or both before combining signals from multiple bands. The feature list alone does not establish which band represents which isoform.
Why might VAPA appear near 33 kDa instead of 27.9 kDa?
Interpretation · The supplied Western blots show an apparent band near 33 kDa, while the predicted mass is 27.9 kDa. VAPA has an alternative isoform and several modified residues, but these features alone do not establish the cause of the difference. Use the observed position as a guide and verify band identity experimentally.
UniProt describes a disulfide-linked VAPA homodimer and a heterodimer with VAPB. Compare reducing and nonreducing samples when investigating a higher band, and verify its identity before assigning it to either complex. Complex formation does not, by itself, predict a specific apparent mass on the blot.
UniProt describes a disulfide-linked VAPA homodimer and a heterodimer with VAPB. Compare reducing and nonreducing samples when investigating a higher band, and verify its identity before assigning it to either complex. Complex formation does not, by itself, predict a specific apparent mass on the blot.
Boster reagents
VAPA 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-VAPA Antibody Picoband®
Cat # A05329-1
The catalog reports one anti-VAPA antibody, A05329-1, for Western blotting, with stated human, mouse, and rat reactivity. Its WB image uses human cell lysates and rat and mouse brain lysates; the reported band is approximately 33 kDa versus an expected 28 kDa.
Which to pick: A05329-1 is the only listed option and has a WB image. Its caption documents human cell lines and rat and mouse brain under reducing conditions. Consider the reported 33 kDa band relative to the expected 28 kDa when interpreting results.
Source: BosterBio VAPA gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9P0L0.
- Human Protein Atlas. VAPA tissue expression.
- PMC5857304 — target-verified WB comparison
- PMC11981147 — target-verified WB comparison
- PMC2878928 — target-verified WB comparison