MYOD1 / Myoblast determination protein 1 · Western blot design guide

Design a Western Blot for MYOD1

Real validated MYOD1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MYOD1 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

Western blot protocol sheet for MYOD1: expected band ~34.5 kDa, hero antibody M00964-3, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **MYOD1** Western blot protocol sheet — expected band ~34.5 kDa, antibody M00964-3, controls and PMC citations. Open the full MYOD1 WB guide →

Navigate to: Protocols Band Controls Antibodies

MYOD1 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	~34.5 kDa
	Observed band	~35 kDa
	Gel	10% (catalog M00964-3)
	Negative control ⓘ	Suggested KO / knockdown lysate

Important caveats

Reasons your observed band may differ from the expected size.

	PTM	Phosphorylated + Acetylated
	Caveat	Modification-state controls
	Gene-set association	MSigDB C7 membership
	Isoform	1 isoform(s)

Section 1

Real Curated MYOD1 Western Blot Protocols

The M00964-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.

Recommended Western blot protocol parameters
Sample / lysate	human PC-3 (catalog M00964-3)
Gel %	10% (catalog M00964-3)
Load	30 ug; reducing conditions (catalog M00964-3)
Transfer	a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M00964-3)
Membrane	nitrocellulose membrane (catalog M00964-3)
Blocking	5% non-fat milk/TBS for 1.5 hour at RT (catalog M00964-3)
Primary antibody	M00964-3 · 1:500 (catalog M00964-3)
Primary incubation	overnight at 4°C (catalog M00964-3)
Secondary antibody	goat anti-rabbit IgG-HRP, 1:5000 (catalog M00964-3)
Secondary incubation	1.5 hour at RT (catalog M00964-3)
Wash	TBS-0.1%Tween 3 times with 5 minutes each (catalog M00964-3)
Detection	ECL (catalog M00964-3)

Section 2

What Is the Expected MYOD1 Western Blot Band Size?

MYOD1 is predicted at 34.5 kDa and observed at ~35 kDa; the small difference has no established cause in the supplied evidence.

What am I looking at on my blot?
Band at ~35 kDa	Consistent with MYOD1; confirm identity with antibody controls.
Faint band at ~35 kDa in whole-cell lysate	Nuclear MYOD1 may be easier to detect after nuclear enrichment.
No band at ~35 kDa in whole-cell lysate	Nuclear extraction or detection may be insufficient.
Additional bands beside ~35 kDa	Their identities are unestablished; annotated modifications do not assign them.

💡Expected MYOD1 appearanceMYOD1 has a predicted mass of 34.5 kDa and an observed band at ~35 kDa; confirm band identity with appropriate antibody controls.

How each factor affects band size
Predicted MYOD1 mass	34.5 kDa predicted; the observed band is ~35 kDa.
Lys104 N6-methylation by EHMT2	Its effect on apparent band size is not established.
Phosphoprotein annotation	No migration change is demonstrated by the supplied evidence.
Ubl conjugation annotation	No conjugate size or distinct band is established.

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	Nuclear MYOD1 may be poorly recovered.	Check nuclear enrichment and extraction.
Band higher than expected	The identity of the higher band is unestablished.	Check antibody specificity against a MYOD1-positive control.
Band lower than expected	The identity of the lower band is unestablished.	Compare with a MYOD1-positive control and check sample integrity.
Multiple bands	Annotated modifications do not establish the identities of extra bands.	Compare bands with a MYOD1-positive control and an independent antibody.
Weak or no signal	Nuclear MYOD1 may be dilute in whole-cell lysate.	Test a nuclear fraction and a MYOD1-positive control.
Fragments below expected size	Sample degradation is possible; no MYOD1 cleavage product is specified.	Check sample handling and compare with freshly prepared lysate.

Source: UniProt P15172 Human Protein Atlas product datasheet (M00964-3)

Experiment evidence

Western blot images showing MYOD1 band

M00964-3 · MYOD1

A00964-2 · MYOD1

M00964-1 · MYOD1

← swipe / scroll for more images →

Real WB data behind the band-size expectations in this section.

Sample controls for MYOD1 Western blot

🧪For positive controls for MYOD1 in Western blot, you can use a validated positive sample once identified; the supplied HPA evidence names none.

Positive control: No high/medium HPA tissue identified

Negative control: Suggested KO / knockdown lysate

Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.

⚠️Feasibility: HPA provides no expression data, so suitable positive and negative samples require validation.

HPA tissue expression evidence for MYOD1

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
No high/medium HPA tissues identified in the supplied evidence.

Lower expression tissues · IHC evidence, not confirmed WB-negative controls

Tissue	Cell type	Level	Evidence	Source
No lower-expression tissue rows available in the supplied evidence.

Section 3

Advanced MYOD1 Western Blot Tips

Deeper troubleshooting and optimisation questions for MYOD1, answered from its protein features.

How should MYOD1 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.

Should I expect multiple MYOD1 isoform bands?

Isoforms · The supplied record lists one isoform and no alternative sequence. It therefore provides no basis for assigning multiple bands to MYOD1 isoforms.

Do MYOD1 modification keywords explain extra bands?

PTM · UniProt keywords include acetylation, phosphorylation, and Ubl conjugation, but the supplied features give no sites for these modifications. Do not assign an extra band to one of them from the keyword alone.

Does this guide establish induction of MYOD1?

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.

How should transfer be checked for MYOD1?

Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.

How should blocking be optimized?

Blocking · Standard workflow guidance: follow the M00964-3 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 I quantify MYOD1 across samples?

Quantitation · MYOD1 is listed as nuclear. Keep sample preparation and the amount of nuclear material compared consistent across samples, then quantify the band near the supplied observed position of about 35 kDa.

Why is the MYOD1 band near 35 kDa?

Interpretation · UniProt predicts 34.5 kDa, close to the supplied observed band of about 35 kDa. This agreement does not establish which features determine its apparent mobility.

UniProt lists N6-methyllysine at position 104, added by EHMT2. This feature alone does not establish a visible band shift. Position 104 uses the supplied UniProt sequence numbering; check the numbering convention before comparing it with an antibody or paper.

Efficient DNA binding requires dimerization with another bHLH protein, and MYOD1 seems to form active heterodimers with ITF-2. That interaction does not establish that a higher band on a Western blot is a dimer; verify the band's identity independently.

Boster reagents

MYOD1 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

Western blot analysis of MYOD using anti-MYOD antibody (M00964-3). Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human PC-3 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-MYOD antigen affinity purified monoclonal antibody (M00964-3) at 1:500 overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for MYOD at approximately 35 kDa. The expected band size for MYOD is at 35 kDa.

Anti-MyoD Rabbit Monoclonal Antibody

Cat # M00964-3

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Real WB data

Western blot analysis of MYOD1 using anti-MYOD1 antibody (A00964-2). Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human RD whole cell lysates, Lane 2: rat heart tissue lysates, Lane 3: rat skeletal muscle tissue lysates, Lane 4: mouse heart tissue lysates, Lane 5: mouse skeletal muscle tissue lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-MYOD1 antigen affinity purified polyclonal antibody (A00964-2) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for MYOD1 at approximately 35-45 kDa. The expected band size for MYOD1 is at 35 kDa.

Anti-MYOD1 Antibody Picoband®

Cat # A00964-2

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Real WB data Western blot analysis of MyoD1 expression in HeLa cell lysate.

Anti-MyoD1/Myod Rabbit Monoclonal Antibody

Cat # M00964-1

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Three the supplier anti-MYOD1 antibodies have Western blot images. M00964-3 was tested in human PC-3 lysate, A00964-2 in human RD and rat and mouse tissue lysates, and M00964-1 in HeLa lysate. These examples document specific tested samples, not universal performance.

Which to pick: For human, mouse, or rat samples, consider A00964-2, whose WB image includes RD cells and rat and mouse heart and skeletal muscle. For human samples, M00964-3 has a detailed PC-3 blot; M00964-1 has a HeLa blot with fewer reported conditions.

Source: BosterBio MYOD1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.

References

UniProt Consortium. UniProt entry P15172.
Human Protein Atlas. MYOD1 tissue expression.
PMC13396186 — target-verified WB comparison
PMC7815108 — target-verified WB comparison
PMC13089754 — target-verified WB comparison