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- Table of Contents
LGMN / Legumain · Western blot design guide
Design a Western Blot for LGMN
Source-linked LGMN Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-LGMN 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
LGMN 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 | ~49.4 kDa | |
| Observed band | ~36, 46, 56 kDa | |
| Gel | 10% (catalog M06476) | |
| Positive control | Placenta (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 | Glycosylated + Cleaved | |
| Caveat | Processing-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked LGMN Western Blot Protocol Options
The M06476 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human 293T, human K562, human MCF-7, rat kidney, rat brain, mouse kidney, mouse brain (catalog M06476) |
| Gel % | 10% (catalog M06476) |
| Load | 30 ug; reducing conditions (catalog M06476) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M06476) |
| Membrane | nitrocellulose membrane (catalog M06476) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M06476) |
| Primary antibody | M06476 · 1:500 (catalog M06476) |
| Primary incubation | overnight at 4°C (catalog M06476) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M06476) |
| Secondary incubation | 1.5 hour at RT (catalog M06476) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M06476) |
| Detection | ECL (catalog M06476) |
Section 2
What Is the Expected LGMN Western Blot Band Size?
LGMN is predicted at 49.4 kDa, while Western blots report ~36, 46, and 56 kDa; the cause of these differences is not established.
| Bands near 36, 46, and 56 kDa | Empirically reported for LGMN; the identity of each band is unassigned |
| Band near 49.4 kDa | Near the predicted mass of the full-length precursor |
| Band below the precursor size | Could reflect signal-peptide or propeptide cleavage |
| Band above the precursor size | Could reflect N-linked glycosylation; the shift is unproven |
| Band near twice the monomer size | Could reflect the precursor homodimer if it survives sample preparation |
💡Expected LGMN appearanceUniProt predicts a 49.4 kDa precursor, while reducing Western blots report ~36, 46, and 56 kDa bands; their identities require antibody-specific validation and band-identity controls.
| Predicted full-length precursor mass | 49.4 kDa before processing or modification |
| N-linked glycosylation at Asn91, Asn167, Asn263, and Asn272 | May increase apparent size; the shift is not established |
| Signal peptide at residues 1–17 | Cleavage may make the mature protein smaller than the precursor |
| Propeptide at residues 324–433 | Autocatalytic removal may yield a smaller processed form |
| Preprocessing homodimer | May appear near twice the monomer size if preserved during electrophoresis |
| Isoforms 1, 2, and 3 | May differ in size; their relative masses and band separation are unknown |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Lysosomal LGMN may be underrepresented in the preparation | Check a positive lysate and assess lysosomal protein recovery |
| Band higher than expected | N-linked glycosylation or a preserved precursor homodimer is possible | Compare reducing conditions and assess glycosylation with appropriate controls |
| Band lower than expected | Signal-peptide or propeptide cleavage is possible | Compare precursor and processed forms with antibodies to defined regions |
| Broad smear instead of sharp band | Heterogeneous N-linked glycosylation is possible but unconfirmed | Compare untreated and deglycosylated samples with controls |
| Multiple bands | The reported bands may include processed forms or isoforms, but assignments are unproven | Use antibody-specific controls and compare antibodies to distinct regions |
| Weak or no signal | The antibody may poorly recognize the LGMN form present in the sample | Verify antibody specificity and test a positive lysate |
| Fragments below expected size | LGMN processing or sample proteolysis may generate smaller species | Add protease inhibitors during preparation and compare antibodies to distinct regions |
Experiment evidence
Western blot images showing LGMN band
M06476 · LGMN
M06476 · LGMN
A06476 · LGMN
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for LGMN Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for LGMN in Western blot, you can use placenta tissue lysate.
Positive control: Placenta (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: LGMN is lysosomal, so use tissue lysate and verify that adipose tissue gives minimal signal.
HPA tissue expression evidence for LGMN
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 |
|---|---|---|---|---|
| Placenta | trophoblastic cells | High | Protein (IHC) | HPA → |
| Skin | fibrohistiocytic cells | High | Protein (IHC) | HPA → |
| Small intestine | enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | neuropil | 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 → |
| Appendix | endocrine cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Hippocampus | glial cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | fibroblasts | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced LGMN Western Blot Tips
Deeper troubleshooting and optimisation questions for LGMN, answered from its protein features.
How should LGMN 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 LGMN isoforms produce different bands?
Isoforms · UniProt lists three isoforms. Isoform 2 lacks 341..397; isoform 3 has a replacement at 341..372 and lacks 373..433. These UniProt canonical coordinates overlap the 324..433 propeptide. Isoform expression and antibody recognition must be established before assigning any observed band to an isoform.
Where are LGMN's listed glycosylation sites?
PTM · UniProt lists N-linked glycosylation at Asn91, Asn167, Asn263, and Asn272, using canonical sequence numbering. These sites may matter when assessing mobility, but their presence alone does not demonstrate a visible shift or identify a particular band.
Does this guide establish induction of LGMN?
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 LGMN?
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 M06476 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 multiple LGMN bands be quantified?
Quantitation · Define which band or bands are being measured and apply that choice consistently across samples. LGMN has precursor processing, glycosylation sites, and three isoforms, so combining all detected bands could mix distinct species. Confirm that the antibody recognizes the species chosen for quantitation.
Why might LGMN bands differ from its predicted mass?
Interpretation · The 49.4 kDa prediction describes full-length LGMN. UniProt lists a signal peptide at 1..17, a propeptide at 324..433 that is removed autocatalytically, and four N-linked glycosylation sites. These features can affect which species are detected, but alone do not establish why the reported bands appear near 36, 46, and 56 kDa.
LGMN is a zymogen with a propeptide at UniProt positions 324..433. UniProt reports autocatalytic propeptide removal, with a homodimer before processing and a monomer afterward. Consider precursor and processed forms when evaluating multiple bands; these features do not uniquely assign the reported 36, 46, or 56 kDa bands.
UniProt lists two disulfide bonds, but the supplied features give no bond coordinates. Reduction could alter migration or antibody recognition, so compare matched reducing conditions if a band is unexpected. Disulfide presence alone cannot establish the identity or apparent mass of a band.
UniProt places LGMN in lysosomes. Consider whether the sampled material contains the lysosomal compartment when interpreting a weak signal. This location supports checking sample composition; it does not predict a specific band intensity or apparent mass.
LGMN is a zymogen with a propeptide at UniProt positions 324..433. UniProt reports autocatalytic propeptide removal, with a homodimer before processing and a monomer afterward. Consider precursor and processed forms when evaluating multiple bands; these features do not uniquely assign the reported 36, 46, or 56 kDa bands.
UniProt lists two disulfide bonds, but the supplied features give no bond coordinates. Reduction could alter migration or antibody recognition, so compare matched reducing conditions if a band is unexpected. Disulfide presence alone cannot establish the identity or apparent mass of a band.
UniProt places LGMN in lysosomes. Consider whether the sampled material contains the lysosomal compartment when interpreting a weak signal. This location supports checking sample composition; it does not predict a specific band intensity or apparent mass.
Boster reagents
LGMN 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-Legumain Rabbit Monoclonal Antibody
Cat # M06476
Real WB data
Anti-Legumain LGMN Antibody
Cat # A06476
Two the supplier anti-LGMN antibodies list Human, Mouse, and Rat reactivity and include WB images. M06476 shows bands near 36, 46, and 56 kDa across specified lysates; A06476 shows SH-SY5Y lysates. These captions do not establish independent validation.
Which to pick: Choose M06476 if you want documented WB conditions and examples from human cells and rat and mouse tissues. A06476 has a WB image from SH-SY5Y lysates at 1:1000, but its caption provides fewer details. Both list Human, Mouse, and Rat reactivity.
Source: BosterBio LGMN gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q99538.
- Human Protein Atlas. LGMN tissue expression.
- PMC13271614 — target-verified WB comparison
- PMC7378441 — target-verified WB comparison
- PMC3158841 — target-verified WB comparison