FMOD / Fibromodulin · Western blot design guide

Design a Western Blot for FMOD

Source-linked FMOD Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-FMOD 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 FMOD: expected band ~43.2 kDa, hero antibody A05030-1, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **FMOD** Western blot protocol sheet — expected band ~43.2 kDa, antibody A05030-1, controls and PMC citations. Open the full FMOD WB guide →

Navigate to: Protocols Band Controls Antibodies

FMOD 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	~43.2 kDa
	Observed band	~60–70 kDa
	Gel	10% (catalog A05030-1)
	Negative control ⓘ	Suggested KO / knockdown lysate

Important caveats

Reasons your observed band may differ from the expected size.

	PTM	Glycosylated + Cleaved
	Caveat	Glycosylation-state controls
	Gene-set association	MSigDB Hallmark membership
	Isoform	1 isoform(s)

Section 1

Source-Linked FMOD Western Blot Protocol Options

The A05030-1 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 HepG2, human Hela, human A431, mouse liver (catalog A05030-1)
Gel %	10% (catalog A05030-1)
Load	30 ug; reducing conditions (catalog A05030-1)
Transfer	a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A05030-1)
Membrane	nitrocellulose membrane (catalog A05030-1)
Blocking	5% non-fat milk/TBS for 1.5 hour at RT (catalog A05030-1)
Primary antibody	A05030-1 · 0.5 μg/mL (catalog A05030-1)
Primary incubation	overnight at 4°C (catalog A05030-1)
Secondary antibody	goat anti-rabbit IgG-HRP, 1:5000 (catalog A05030-1)
Secondary incubation	1.5 hour at RT (catalog A05030-1)
Wash	TBS-0.1%Tween 3 times with 5 minutes each (catalog A05030-1)
Detection	ECL (catalog A05030-1)

Section 2

What Is the Expected FMOD Western Blot Band Size?

FMOD's predicted precursor is 43.2 kDa and its observed band is ~60–70 kDa; the cause of this difference is not established.

What am I looking at on my blot?
Band at ~60–70 kDa	Empirical FMOD-reactive band; its migration above the predicted mass is unexplained.
Band near 43 kDa	Near the sequence-predicted precursor mass; confirm its identity.
Band below the precursor position	Could reflect cleavage of the 1–18 signal peptide; confirm its identity.
Broad band or smear	Could reflect variation in N-linked keratan sulfate modification; a visible effect is unproven.
Little or no band in whole-cell lysate	FMOD is secreted into the extracellular matrix.

💡Expected FMOD appearanceFMOD has a predicted 43.2 kDa precursor mass, while antibody QC reports a ~60–70 kDa band in reducing lysates; the cause of the difference is unproven, so confirm band identity with appropriate controls.

How each factor affects band size
Predicted 43.2 kDa precursor mass	Provides a sequence-based baseline; the empirical band is ~60–70 kDa.
Signal peptide at residues 1–18	Cleavage makes mature FMOD smaller than its precursor.
N-linked keratan sulfate site at Asn127	Modification could alter apparent migration; its contribution is unmeasured.
N-linked keratan sulfate site at Asn166	Modification could alter apparent migration; its contribution is unmeasured.
N-linked keratan sulfate site at Asn201	Modification could alter apparent migration; its contribution is unmeasured.
N-linked keratan sulfate site at Asn291	Modification could alter apparent migration; its contribution is unmeasured.
N-linked glycosylation site at Asn341	Modification could alter apparent migration; its contribution is unmeasured.

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	FMOD is secreted into the extracellular matrix.	Check an extracellular matrix-enriched sample alongside a positive control.
Band higher than expected	The empirical ~60–70 kDa band exceeds the 43.2 kDa precursor prediction; the cause is unproven.	Compare with a positive control and verify identity using an independent antibody.
Band lower than expected	Signal peptide cleavage can make mature FMOD smaller than its precursor.	Check sample integrity and confirm the band with an independent antibody.
Broad smear instead of sharp band	Variable modification at the N-linked glycosylation sites is possible.	Compare untreated and glycan-removal-treated samples with a positive control.
Multiple bands	Different glycosylation or processing states are possible, but distinct bands are not established.	Compare glycan-removal-treated samples and verify bands with an independent antibody.
Weak or no signal	The sampled fraction may contain little secreted FMOD.	Check loading and antibody performance with a positive control, then test an extracellular matrix-enriched sample.

Source: UniProt Q06828 Human Protein Atlas product datasheet (A05030-1)

Experiment evidence

Western blot images showing FMOD band

A05030-1 · FMOD

← swipe / scroll for more images →

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

Sample controls for FMOD Western blot

🧪For positive controls for FMOD in Western blot, you can use an HPA-IHC candidate positive sample once one is identified.

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 such as Ponceau alongside the samples.

⚠️Feasibility: Secreted FMOD may be sparse in whole-cell lysate, so conditioned medium may be preferable.

HPA tissue expression evidence for FMOD

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 FMOD Western Blot Tips

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

How should FMOD 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.

Do multiple FMOD bands indicate isoforms?

Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. Multiple bands therefore cannot be assigned to listed isoforms from this evidence. Consider the annotated glycosylation and other modifications as possibilities, then verify band identity experimentally before interpreting each band as FMOD.

Which FMOD glycosylation sites matter when interpreting band patterns?

PTM · UniProt lists N-linked sites at residues 127, 166, 201, 291, and 341. The first four are annotated with keratan sulfate. Compare bands across consistently prepared samples, and use a validated glycan-removal control if testing whether glycosylation contributes to their migration.

Does this guide establish induction of FMOD?

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 FMOD?

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 A05030-1 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 FMOD bands be quantified?

Quantitation · Define the band or band region consistently across samples and report which signal was measured. FMOD has a reported apparent band near 60–70 kDa, but its glycosylation features alone do not identify every nearby band. Keep unexpected bands separate until their identity is established.

Why might FMOD migrate above its predicted 43.2 kDa?

Interpretation · An apparent band near 60–70 kDa is reported. FMOD has five listed N-linked glycosylation sites, four associated with keratan sulfate, which could affect migration. The features alone do not establish how much of the difference glycosylation explains.

UniProt lists sulfotyrosines at residues 20, 38, 39, 45, 47, 53, and 55. These coordinates use UniProt numbering. Their presence does not establish a visible band shift, so do not assign an unexpected band to sulfation without experimental evidence.

The listed signal peptide spans UniProt residues 1–18, and residue 19 is annotated as pyrrolidone carboxylic acid. Account for this processing when comparing sequence-based mass with a blot band; it does not by itself explain the reported 60–70 kDa apparent mass.

FMOD is annotated as secreted and located in the extracellular space and extracellular matrix. Include the relevant extracellular or matrix-containing fraction when assessing its abundance; a cell-only preparation may miss FMOD present outside cells.

Boster reagents

FMOD 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 FMOD using anti-FMOD antibody (A05030-1). 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 HepG2 whole cell lysates, Lane 2: human Hela whole cell lysates, Lane 3: human A431 whole cell lysates, Lane 4: mouse liver 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-FMOD antigen affinity purified polyclonal antibody (A05030-1) 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 FMOD at approximately 60-70 kDa. The expected band size for FMOD is at 43 kDa.

Anti-Fibromodulin/FMOD Antibody Picoband®

Cat # A05030-1

View product →

A05030-1 is a rabbit polyclonal anti-FMOD antibody listed for human and mouse Western blots. Its WB image uses human HepG2, HeLa and A431 cell lysates and mouse liver lysate. The reported band is 60–70 kDa, versus an expected 43 kDa.

Which to pick: A05030-1 is the only listed option. Its WB image includes human cell and mouse liver samples, so compare your specimen with those contexts and account for the reported 60–70 kDa band when interpreting results.

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

References

UniProt Consortium. UniProt entry Q06828.
Human Protein Atlas. FMOD tissue expression.
PMC8393414 — target-verified WB comparison
PMC11891772 — target-verified WB comparison
PMC6697857 — target-verified WB comparison