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- Table of Contents
MYH9 / Myosin-9 · Western blot design guide
Design a Western Blot for MYH9
Real validated MYH9 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MYH9 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
MYH9 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 | ~226.5 kDa | |
| Observed band | ~220–250 kDa | |
| Gel | 8% (catalog A00880-2) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Caudate (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 Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated MYH9 Western Blot Protocols
The A00880-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human A549, human U2OS, human U251 (catalog A00880-2) |
| Gel % | 8% (catalog A00880-2) |
| Load | 30 ug; reducing conditions (catalog A00880-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00880-2) |
| Membrane | nitrocellulose membrane (catalog A00880-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00880-2) |
| Primary antibody | A00880-2 · 0.5 μg/mL (catalog A00880-2) |
| Primary incubation | overnight at 4°C (catalog A00880-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00880-2) |
| Secondary incubation | 1.5 hour at RT (catalog A00880-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00880-2) |
| Detection | ECL (catalog A00880-2) |
Section 2
What Is the Expected MYH9 Western Blot Band Size?
MYH9 is predicted at 226.5 kDa and observed at ~220–250 kDa; the cause of any migration difference is not established.
| Band at ~220–250 kDa | Empirical MYH9 band, consistent with its 226.5 kDa predicted mass |
| Band above ~250 kDa | May reflect incompletely dissociated MYH9 heavy-chain assembly |
| Several bands near the expected region | Could reflect isoforms 1 and 2 if their migration differs |
| Weak band in a soluble lysate fraction | Cytoskeletal MYH9 may be underrepresented |
💡Expected MYH9 appearanceMYH9 has a predicted mass of 226.5 kDa and an empirical band at ~220–250 kDa; confirm band identity with appropriate antibody and sample controls.
| Predicted MYH9 mass | The full-length chain is 226.5 kDa; the empirical band is ~220–250 kDa |
| Two heavy chains in the myosin complex | Incomplete dissociation may retain higher-mass material; no inter-chain disulfide is listed |
| Splice isoform 1 | Its size relative to isoform 2 is not supplied |
| Splice isoform 2 | Its size relative to isoform 1 is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Cytoskeletal MYH9 may be poorly recovered | Check the insoluble fraction and use a known positive lysate |
| Band higher than expected | The heavy-chain assembly may be incompletely dissociated | Check denaturation conditions and compare with a fully denatured sample |
| Band lower than expected | An alternative isoform or an unrelated band is possible; its size is unreported | Confirm identity with an antibody to another MYH9 epitope |
| Multiple bands | Isoforms 1 and 2 may differ in migration, though resolution is unproven | Compare bands using antibodies to distinct MYH9 epitopes |
| Weak or no signal | Cytoskeletal MYH9 may be underrepresented in the tested fraction | Check fraction recovery and include a positive lysate |
| Fragments below expected size | The long MYH9 heavy chain may have degraded during sample preparation | Prepare fresh lysate with protease inhibitors and verify fragments with another epitope |
Experiment evidence
Western blot images showing MYH9 band
A00880-2 · MYH9
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for MYH9 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for MYH9 in Western blot, you can use appendix tissue lysate, which HPA rates High.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Caudate (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside MYH9.
⚠️Feasibility: A cytoskeletal MYH9 signal should be accessible in tissue lysate, but validate caudate as a negative control.
HPA tissue expression evidence for MYH9
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 → |
| Fallopian tube | glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | glandular cells | High | Protein (IHC) | HPA → |
| Kidney | cells in glomeruli | High | Protein (IHC) | HPA → |
| Liver | cholangiocytes | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Hippocampus | glial cells | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MYH9 Western Blot Tips
Deeper troubleshooting and optimisation questions for MYH9, answered from its protein features.
How should MYH9 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 MYH9 isoform 2 produce a smaller band?
Isoforms · Yes. Isoform 2 lacks canonical residues 1–136 and 980–1421, so a substantially smaller product is plausible. These are canonical UniProt coordinates; compare the antibody’s recognized region with the missing segments before assigning a band to isoform 2.
Which MYH9 phosphorylation sites can guide phospho-specific detection?
PTM · UniProt lists phosphotyrosine at 11 and 754 and phosphoserine at 628, 1114, 1714, and 1943. These are canonical UniProt coordinates; check the antibody’s numbering and target site. Listed sites do not establish that phosphorylation produces a resolvable band shift.
MYH9 has listed acetyllysine, succinyllysine, and methylarginine modifications, including succinyllysine at canonical UniProt positions 850 and 1669 and methylarginine at 1923. Their presence alone cannot identify an extra band or demonstrate a visible mobility shift.
MYH9 has listed acetyllysine, succinyllysine, and methylarginine modifications, including succinyllysine at canonical UniProt positions 850 and 1669 and methylarginine at 1923. Their presence alone cannot identify an extra band or demonstrate a visible mobility shift.
Does this guide establish induction of MYH9?
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 MYH9 Western blot?
Transfer · Choose and validate transfer conditions for the approximately 226.5 kDa MYH9 heavy chain. Check the membrane and the gel after transfer to see whether protein of this size transferred adequately; the supplied features do not specify one transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A00880-2 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 MYH9 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.
Why does MYH9 appear around 220–250 kDa?
Interpretation · The supplied apparent band range includes MYH9’s predicted mass of 226.5 kDa. Its listed modifications do not, by themselves, establish a visible shift or explain a difference between predicted and apparent mass.
Compare the same band and sample fraction across lanes. MYH9 is listed in the cytoskeleton, cell cortex, vesicles, and cell membrane, so differences in fraction preparation can affect the measured signal. Keep loading and detection conditions consistent.
Consider the two isoforms and the regions missing from isoform 2, then check whether the antibody recognizes a retained region. MYH9 also forms a complex with light chains, but the supplied features do not identify any particular unexpected band. A listed modification alone is insufficient to assign its identity.
Compare the same band and sample fraction across lanes. MYH9 is listed in the cytoskeleton, cell cortex, vesicles, and cell membrane, so differences in fraction preparation can affect the measured signal. Keep loading and detection conditions consistent.
Consider the two isoforms and the regions missing from isoform 2, then check whether the antibody recognizes a retained region. MYH9 also forms a complex with light chains, but the supplied features do not identify any particular unexpected band. A listed modification alone is insufficient to assign its identity.
Boster reagents
MYH9 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-MYH9 Antibody Picoband®
Cat # A00880-2
The catalog reports one anti-MYH9 antibody, A00880-2, with a Western blot image showing an approximately 220–250 kDa band (expected: 227 kDa). The caption documents human cell lines and rat and mouse tissue lysates; it does not provide orthogonal validation.
Which to pick: A00880-2 is the only listed option. Its reported reactivity is human, mouse and rat, and its WB image includes human A549, U2OS and U251 cells plus rat and mouse kidney and brain lysates under reducing conditions.
Source: BosterBio MYH9 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P35579.
- Human Protein Atlas. MYH9 tissue expression.
- PMC8923078 — target-verified WB comparison
- PMC7018736 — target-verified WB comparison
- PMC11873648 — target-verified WB comparison