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- Table of Contents
MYL3 / Myosin light chain 3 · Western blot design guide
Design a Western Blot for MYL3
Source-linked MYL3 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MYL3 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
MYL3 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 | ~21.9 kDa | |
| Observed band | ~25 kDa | |
| Gel | 5–20% (catalog A04730-2) | |
| Positive control | Heart muscle (IHC candidate; verify WB) +1 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Methylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked MYL3 Western Blot Protocol Options
The A04730-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Caco-2, human Hela, human H9C2(2-1), mouse heart, mouse skeletal muscle, mouse C2C12 (catalog A04730-2) |
| Gel % | 5–20% (catalog A04730-2) |
| Load | 30 ug; reducing conditions (catalog A04730-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A04730-2) |
| Membrane | nitrocellulose membrane (catalog A04730-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A04730-2) |
| Primary antibody | A04730-2 · 0.5 μg/mL (catalog A04730-2) |
| Primary incubation | overnight at 4°C (catalog A04730-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A04730-2) |
| Secondary incubation | 1.5 hour at RT (catalog A04730-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A04730-2) |
| Detection | ECL (catalog A04730-2) |
Section 2
What Is the Expected MYL3 Western Blot Band Size?
MYL3 is predicted at 21.9 kDa and observed at ~25 kDa; the cause of the difference is not established.
| Band at ~25 kDa | Empirical MYL3 band reported in antibody QC; its identity still warrants a positive control. |
| Band near 21.9 kDa | Near the UniProt predicted mass; confirm its identity with a positive control. |
| Closely spaced bands near the monomer | MYL3 has phosphorylation sites, but distinct phosphoform bands have not been demonstrated. |
| Weak or absent band in a sample lysate | MYL3 is a muscle protein; compare with a heart lysate positive control. |
💡Expected MYL3 appearanceMYL3 is predicted at 21.9 kDa, while antibody QC reports a band at ~25 kDa; the cause of the difference is unproven, so confirm identity with a positive control and an independent antibody.
| UniProt predicted mass | 21.9 kDa by sequence; antibody QC observes ~25 kDa. |
| N,N,N-trimethylalanine at residue 2 | A recorded modification with no demonstrated visible migration effect. |
| Phosphothreonine at residues 88, 127 and 129 | Phosphorylation may alter mobility; a resolved shift is unproven. |
| Phosphotyrosine at residue 130 | Phosphorylation may alter mobility; a resolved shift is unproven. |
| Phosphoserine at residue 179 | Phosphorylation may alter mobility; a resolved shift is unproven. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | MYL3 signal may be low in the tested sample. | Run a heart lysate positive control alongside it. |
| Band higher than expected | The ~25 kDa empirical band exceeds the 21.9 kDa predicted mass for an unestablished reason. | Compare with the reported ~25 kDa positive control band and confirm with an independent antibody. |
| Band lower than expected | Band identity or sample integrity is uncertain. | Check sample handling and compare with a positive control and an independent antibody. |
| Multiple bands | MYL3 has several phosphorylation sites, although distinct phosphoform bands are unproven. | Compare matched samples with and without phosphatase treatment and verify band identity. |
| Weak or no signal | MYL3 abundance may be low in the tested sample. | Check antibody performance with a heart lysate positive control. |
Experiment evidence
Western blot images showing MYL3 band
A04730-2 · MYL3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for MYL3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for MYL3 in Western blot, you can use heart muscle lysate.
Positive control: Heart muscle (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.
⚠️Feasibility: High expression in heart muscle and no detected expression in adipose tissue make tissue controls feasible.
HPA tissue expression evidence for MYL3
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 |
|---|---|---|---|---|
| Heart muscle | cardiomyocytes | High | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MYL3 Western Blot Tips
Deeper troubleshooting and optimisation questions for MYL3, answered from its protein features.
How should MYL3 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 MYL3 isoforms explain multiple bands?
Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. It therefore provides no isoform basis for assigning multiple bands to MYL3.
Which MYL3 modifications matter when interpreting bands?
PTM · UniProt lists N,N,N-trimethylalanine at position 2; phosphothreonine at 88, 127, and 129; phosphotyrosine at 130; and phosphoserine at 179. These are UniProt coordinates; check the numbering convention used by any antibody or paper. Their presence does not establish a visible band shift.
Specify which listed site the assay measures: threonine 88, 127, or 129; tyrosine 130; or serine 179, using UniProt numbering. Compare that signal with a total MYL3 measurement so a change in phosphorylation is not mistaken for a change in protein abundance.
Specify which listed site the assay measures: threonine 88, 127, or 129; tyrosine 130; or serine 179, using UniProt numbering. Compare that signal with a total MYL3 measurement so a change in phosphorylation is not mistaken for a change in protein abundance.
Does this guide establish induction of MYL3?
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 MYL3 Western blot?
Transfer · MYL3 has a predicted mass of 21.9 kDa. Choose transfer conditions that retain proteins near this size, then verify MYL3 recovery on the membrane with an appropriate marker or control. The supplied features do not specify a particular transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A04730-2 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 MYL3 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 might MYL3 appear near 25 kDa instead of 21.9 kDa?
Interpretation · The supplied blot observation is approximately 25 kDa, while the predicted mass is 21.9 kDa. Compare the band with a molecular weight marker and verify its identity. The listed modifications alone do not establish the cause of this difference.
UniProt lists N,N,N-trimethylalanine at position 2. That annotation identifies a modification, but does not show that methylated MYL3 resolves as a separate band. Confirm any proposed assignment independently.
UniProt describes myosin as a hexamer containing two heavy chains and four light chains. This association gives context for investigating high-mass signal, but does not identify a particular band or establish that the complex survives sample preparation. Verify band identity before assigning it to MYL3.
UniProt lists N,N,N-trimethylalanine at position 2. That annotation identifies a modification, but does not show that methylated MYL3 resolves as a separate band. Confirm any proposed assignment independently.
UniProt describes myosin as a hexamer containing two heavy chains and four light chains. This association gives context for investigating high-mass signal, but does not identify a particular band or establish that the complex survives sample preparation. Verify band identity before assigning it to MYL3.
Boster reagents
MYL3 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-MYL3 Antibody Picoband®
Cat # A04730-2
The catalog reports one anti-MYL3 antibody, A04730-2, with reported human, mouse, and rat reactivity. Its WB image shows a band near 25 kDa (expected 22 kDa). The supplied evidence does not establish specificity through independent validation.
Which to pick: A04730-2 is the only listed option. Its WB caption includes Caco-2, HeLa, H9C2(2-1), mouse heart, mouse skeletal muscle, and C2C12 lysates. Rat reactivity is listed, but the caption does not identify a rat sample.
Source: BosterBio MYL3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P08590.
- Human Protein Atlas. MYL3 tissue expression.
- PMC10909433 — target-verified WB comparison
- PMC12536539 — target-verified WB comparison
- PMC7034401 — target-verified WB comparison