This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
MYL9 / Myosin regulatory light polypeptide 9 · Western blot design guide
Design a Western Blot for MYL9
Real validated MYL9 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MYL9 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
MYL9 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 | ~19.8 kDa | |
| Gel | 15% (standard starting point) | |
| Positive control | Duodenum (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 Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated MYL9 Western Blot Protocols
The M06446-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Human uterus lysate (catalog M06446-1) |
| Gel % | 15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.2 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | M06446-1; 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 MYL9 Western Blot Band Size?
MYL9 is predicted at 19.8 kDa; isoforms and phosphorylation could affect migration, but no empirical band or visible shift is established.
| Band near 19.8 kDa | Consistent with the predicted MYL9 polypeptide; confirm identity with antibody controls |
| Additional band at a different position | Could reflect isoforms 1 and 2; their migration is not established |
| Subtle doublet near the expected band | Could reflect phosphorylation at Thr19 or Ser20; a visible shift is not established |
| Faint band in a soluble lysate fraction | MYL9 associates with the cytoskeleton and may be underrepresented in that fraction |
💡Expected MYL9 appearanceUniProt predicts a 19.8 kDa MYL9 polypeptide; no empirical band size is supplied, and any band assignment needs ordinary identity controls.
| UniProt predicted mass | Sets a 19.8 kDa sequence-based reference, not a measured band position |
| Isoform 1 | Its individual mass and migration are not supplied |
| Isoform 2 | Its individual mass and migration are not supplied |
| Alternative splicing of isoforms 1 and 2 | May change apparent size, but the direction and visibility are unknown |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Cytoskeletal association may limit extraction | Check the insoluble fraction and an appropriate positive lysate |
| Band higher than expected | An isoform or incompletely disrupted myosin association is possible | Check denaturation conditions and confirm identity with an independent antibody |
| Band lower than expected | Isoform migration is unknown; band identity is uncertain | Use an independent antibody and compare a MYL9-depleted sample |
| Multiple bands | Isoforms 1 and 2 or phosphorylation at Thr19 and Ser20 may contribute | Confirm bands with MYL9 depletion and compare phosphatase-treated samples |
| Weak or no signal | Cytoskeletal MYL9 may be poorly recovered in the sampled fraction | Check extraction and compare soluble and insoluble fractions |
Experiment evidence
Western blot images showing MYL9 band
M06446-1 · MYL9
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for MYL9 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for MYL9 in Western blot, you can use duodenum tissue, which shows high HPA expression.
Positive control: Duodenum (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: Tissue controls are feasible, although HPA non-detection in adipose tissue may not guarantee an absent Western-blot signal.
HPA tissue expression evidence for MYL9
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 |
|---|---|---|---|---|
| Duodenum | endocrine cells | High | Protein (IHC) | HPA → |
| Skin | epidermal cells | High | Protein (IHC) | HPA → |
| Small intestine | endocrine cells | High | Protein (IHC) | HPA → |
| Breast | myoepithelial cells | Medium | Protein (IHC) | HPA → |
| Cervix | squamous epithelial 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 → |
| Appendix | endocrine cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MYL9 Western Blot Tips
Deeper troubleshooting and optimisation questions for MYL9, answered from its protein features.
What band size should I expect for MYL9?
Band shift · The supplied predicted mass is 19.8 kDa; no observed band size is available. The annotated modifications do not establish a visible shift or explain a difference between predicted and apparent mass. Compare the band with a molecular weight marker and consider the two annotated isoforms.
Could MYL9 isoforms produce different bands?
Isoforms · Yes. Isoform 2 lacks residues 62–115 of the 172-residue canonical sequence, so it may migrate differently from isoform 1. The features do not establish either isoform’s apparent band position or expression in your sample.
Which MYL9 modifications matter when interpreting bands?
PTM · UniProt annotates N-acetylserine at position 2, phosphothreonine at position 19, and phosphoserine at position 20. These are UniProt coordinates; antibody or paper numbering may differ. Modification sites alone do not demonstrate a visible mobility shift.
At UniProt position 19, the annotated kinases are MLCK, CIT and ROCK2. At position 20, they are CDC42BP, CIT, MLCK, PAK1, ROCK1, ROCK2, DAPK1, DAPK2 and ZIPK/DAPK3. These annotations do not establish how a particular treatment will affect phosphorylation.
For a site-specific measurement, distinguish phosphorylation at UniProt position 19 from position 20 and normalize the site-specific signal to total MYL9 in the same samples. Check antibody numbering against UniProt coordinates. The two annotated isoforms may also affect which bands contribute to total MYL9.
At UniProt position 19, the annotated kinases are MLCK, CIT and ROCK2. At position 20, they are CDC42BP, CIT, MLCK, PAK1, ROCK1, ROCK2, DAPK1, DAPK2 and ZIPK/DAPK3. These annotations do not establish how a particular treatment will affect phosphorylation.
For a site-specific measurement, distinguish phosphorylation at UniProt position 19 from position 20 and normalize the site-specific signal to total MYL9 in the same samples. Check antibody numbering against UniProt coordinates. The two annotated isoforms may also affect which bands contribute to total MYL9.
Does this guide establish induction of MYL9?
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 MYL9 Western blot?
Transfer · MYL9 has a predicted mass of 19.8 kDa, and isoform 2 lacks residues 62–115. Choose transfer settings suited to retaining small proteins and check transfer near the expected size with a marker. The supplied features do not identify one required transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M06446-1 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 MYL9 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.
How should I interpret unexpected MYL9 bands?
Interpretation · First compare them with the 19.8 kDa prediction and consider isoform 2, which lacks residues 62–115. MYL9 also has annotated acetylation and phosphorylation sites, but their presence alone cannot assign an unexpected band or prove a mobility shift. No empirical band position was supplied.
MYL9 is annotated in the cytoskeleton and cell cortex, with colocalization at the actomyosin cortex in myoblasts. When comparing samples, use a consistent extraction procedure and assess whether your preparation retains the cytoskeletal material of interest.
MYL9 is annotated in the cytoskeleton and cell cortex, with colocalization at the actomyosin cortex in myoblasts. When comparing samples, use a consistent extraction procedure and assess whether your preparation retains the cytoskeletal material of interest.
Boster reagents
MYL9 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-MYL9 Rabbit Monoclonal Antibody
Cat # M06446-1
The catalog reports a rabbit monoclonal anti-MYL9 antibody for Western blotting, with reported Human, Mouse, and Rat reactivity. Its supplied WB image shows MYL9 expression in human uterus lysate; image evidence for Mouse and Rat samples is not provided.
Which to pick: M06446-1 is the only listed option. Its WB image uses human uterus lysate, so it has direct image evidence for that sample; check the reported reactivity when planning Mouse or Rat blots.
Source: BosterBio MYL9 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P24844.
- Human Protein Atlas. MYL9 tissue expression.
- PMC11946870 — target-verified WB comparison
- PMC9496379 — target-verified WB comparison
- PMC9795222 — target-verified WB comparison