This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
MYO5A / Unconventional myosin-Va · Western blot design guide
Design a Western Blot for MYO5A
Source-linked MYO5A Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MYO5A 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
MYO5A 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 | ~215.4 kDa | |
| Gel | 4–12% gradient (standard starting point) | |
| Positive control | Cerebellum (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 | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked MYO5A Western Blot Protocol Options
The A02216-1 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 A02216-1) |
| Gel % | 4–12% gradient (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 A02216-1) |
| Primary antibody | A02216-1 · 1:1000 (catalog A02216-1) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Goat Anti-Rabbit IgG, 1:10000 (catalog A02216-1) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL (catalog A02216-1) |
Section 2
What Is the Expected MYO5A Western Blot Band Size?
MYO5A has a predicted monomer mass of 215.4 kDa; isoforms, phosphorylation, and possible homodimerization could affect appearance, but altered migration is unproven.
| Band near 215.4 kDa | Consistent with the predicted MYO5A monomer; confirm identity with controls |
| Band near twice the monomer size | Could reflect the reported possible homodimer if it survives electrophoresis |
| Several bands at different positions | Could reflect isoforms 1, 2, and 3 if their migration differs |
| Close doublet near the monomer position | Could reflect phosphorylation-dependent migration, but this is unproven |
💡Expected MYO5A appearanceUniProt predicts a 215.4 kDa MYO5A monomer; no empirical band size is supplied, so confirm a band near that position with appropriate identity controls.
| Predicted monomer mass | Sets a reference position near 215.4 kDa |
| Possible homodimer | Could appear near twice the monomer size if retained during electrophoresis |
| Splice isoforms 1, 2, and 3 | May migrate differently; their individual sizes are not supplied |
| Phosphorylation at Ser600, Thr1032, Ser1452, Ser1652, and Thr1760 | May alter migration; no visible shift is established |
| N-acetylalanine at residue 2 | Adds a small modification; no visible shift is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | MYO5A may be below detection in the tested lysate | Check a positive-control lysate and loading |
| Band higher than expected | The reported possible homodimer may persist during electrophoresis | Compare denaturing conditions and confirm identity with an independent antibody |
| Band lower than expected | An isoform or sample degradation could account for a smaller band; no size is established | Use fresh lysate and an independent MYO5A antibody |
| Multiple bands | Isoforms 1, 2, and 3 or phosphorylation may contribute, but distinct migration is unproven | Compare bands across samples with an independent antibody |
| Weak or no signal | Low target abundance or insufficient detection | Check loading, antibody performance, and a positive-control lysate |
Experiment evidence
Western blot images showing MYO5A band
A02216-1 · MYO5A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for MYO5A Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for MYO5A in Western blot, you can use cerebellum tissue, which HPA scores as High.
Positive control: Cerebellum (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: HPA reports no detection in adipose tissue, making tissue-based negative controls feasible.
HPA tissue expression evidence for MYO5A
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 |
|---|---|---|---|---|
| Cerebellum | cells in granular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuropil | High | Protein (IHC) | HPA → |
| Appendix | lymphoid tissue | Medium | Protein (IHC) | HPA → |
| Breast | glandular cells | Medium | Protein (IHC) | HPA → |
| Colon | glandular cells | Medium | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MYO5A Western Blot Tips
Deeper troubleshooting and optimisation questions for MYO5A, answered from its protein features.
How should MYO5A 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 MYO5A isoforms produce different bands?
Isoforms · Three isoforms are listed. Relative to isoform 1, isoform 2 lacks residues 1321–1347, while isoform 3 replaces L1413 with LYFEELYADDPKKYQSYRISLYKRMI. These sequence changes could affect band position, but the features do not establish whether the isoforms resolve as separate bands. Check which isoforms the antibody recognizes.
Which MYO5A modifications matter when interpreting bands?
PTM · UniProt lists N-acetylalanine at position 2; phosphoserine at 600, 1452 and 1652; and phosphothreonine at 1032 and 1760. These are UniProt coordinates and may differ from paper or antibody numbering. The listed modifications alone do not establish a visible shift. Match any phospho-specific antibody to its stated site and numbering convention.
Does this guide establish induction of MYO5A?
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 MYO5A Western blot?
Transfer · MYO5A has a predicted mass of 215.4 kDa. A wet transfer is a practical starting point for a protein this large; check transfer efficiency near 215 kDa and adjust transfer conditions if signal remains in the gel.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02216-1 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 MYO5A bands be quantified?
Quantitation · Quantify a band only after checking that its apparent size and antibody recognition are consistent with the MYO5A isoform of interest. The three listed isoforms and five phosphorylation sites make it useful to state whether the measurement targets total MYO5A, a particular isoform or a specific phosphosite.
How should MYO5A band position compare with its predicted mass?
Interpretation · The predicted mass is 215.4 kDa. No observed band position is supplied, so a difference between predicted and apparent mass cannot be established from these features. Compare the band with a high molecular weight marker and report its apparent mass.
Consider the listed splice variants: isoform 2 lacks residues 1321–1347, and isoform 3 has a replacement at 1413. MYO5A may also form a homodimer and associate with calmodulin or myosin light chains, although these features do not establish that such complexes survive the blot conditions. A band cannot be assigned to an isoform, modification or complex from position alone.
Consider the listed splice variants: isoform 2 lacks residues 1321–1347, and isoform 3 has a replacement at 1413. MYO5A may also form a homodimer and associate with calmodulin or myosin light chains, although these features do not establish that such complexes survive the blot conditions. A band cannot be assigned to an isoform, modification or complex from position alone.
Boster reagents
MYO5A 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-Unconventional myosin-Va MYO5A Antibody
Cat # A02216-1
A02216-1 is listed for human, mouse, and rat MYO5A. Its WB image shows extracts from various cell lines tested at 1:1000 with 25 µg per lane; the caption does not identify the cell lines or establish which species were tested.
Which to pick: A02216-1 is the only listed MYO5A antibody and has a WB image. Use the reported 1:1000 dilution as a starting point, and verify performance in your own sample.
Source: BosterBio MYO5A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9Y4I1.
- Human Protein Atlas. MYO5A tissue expression.
- PMC6016585 — target-verified WB comparison
- PMC9851619 — target-verified WB comparison
- PMC10740236 — target-verified WB comparison