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- Table of Contents
LAG3 / Lymphocyte activation gene 3 protein · Western blot design guide
Design a Western Blot for LAG3
Real validated LAG3 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-LAG3 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
LAG3 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 | ~57.4 kDa | |
| Gel | 12–15% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated LAG3 Western Blot Protocols
The A02869 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | mouse liver tissue lysate (catalog A02869) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A02869 · 1 μg/mL (catalog A02869) |
| 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 LAG3 Western Blot Band Size?
LAG3 has a predicted 57.4 kDa precursor; cleavage, N-linked glycosylation and homodimerization could affect migration, but no empirical band position is supplied.
| Band near 57.4 kDa | Candidate precursor-sized LAG3; confirm its identity with antibody controls |
| Band above 57.4 kDa | N-linked glycosylation at Asn188, Asn250, Asn256 or Asn343 could affect migration; no visible shift is established |
| Band below 57.4 kDa | Signal-peptide cleavage could yield a smaller mature form |
| Band near twice the monomer size | Could reflect a retained LAG3 homodimer; an inter-chain disulfide is not documented |
| Several bands at different positions | Could include isoforms 1 and 2 if their migration differs; distinct bands are not established |
💡Expected LAG3 appearanceUniProt predicts a 57.4 kDa LAG3 precursor; signal-peptide cleavage, N-linked glycosylation and homodimerization may affect its blot pattern, but no empirical band position is supplied, so confirm identity with antibody controls.
| 57.4 kDa predicted precursor mass | Provides the reference size, not a validated blot position |
| N-linked glycosylation at Asn188, Asn250, Asn256 and Asn343 | May increase apparent size; the magnitude and visibility are unknown |
| Signal peptide at residues 1–22 | Its cleavage makes the mature chain smaller than the precursor |
| LAG3 homodimer | Could yield a band near twice the monomer size if retained during electrophoresis |
| Isoforms 1 and 2 | May differ in size, but their relative masses and band separation are unknown |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-bound LAG3 may be poorly recovered | Check membrane extraction and a LAG3-positive control |
| Band higher than expected | N-linked glycosylation or a retained homodimer may affect migration | Compare glycosidase-treated and untreated samples, and reducing and nonreducing lanes |
| Band lower than expected | Signal-peptide cleavage or an isoform may yield a smaller product | Check antibody epitope coverage and confirm identity with a second antibody |
| Broad smear instead of sharp band | Heterogeneous N-linked glycosylation is possible but unproven | Compare glycosidase-treated and untreated samples with a positive control |
| Multiple bands | Isoforms 1 and 2 or different processing states may contribute | Check antibody specificity with an independent antibody or LAG3-deficient control |
| Weak or no signal | Membrane extraction or antibody recognition may be inadequate | Verify sample recovery and antibody performance with a LAG3-positive control |
Experiment evidence
Western blot images showing LAG3 band
A02869 · LAG3
M02869-1 · LAG3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for LAG3 Western blot
🧪For positive controls for LAG3 in Western blot, you can use no specific sample identified by the supplied HPA evidence.
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: LAG3 is a membrane protein, but the supplied HPA data identify no positive sample, so a positive control needs separate validation.
HPA tissue expression evidence for LAG3
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 LAG3 Western Blot Tips
Deeper troubleshooting and optimisation questions for LAG3, answered from its protein features.
How should LAG3 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 LAG3 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. In isoform 2, residues 353–360 change from VTPKSFGS to GQPQVGKE, and residues 361–525 are missing. Check whether the antibody recognizes a region retained in both isoforms before assigning or comparing bands. These are UniProt sequence coordinates.
An antibody recognizing residues 361–525 cannot detect isoform 2, which lacks that region. For comparisons across samples, establish which isoform or bands the antibody measures and quantify the same bands consistently. Glycosylation at the four listed sites may also complicate assignment of a single band.
An antibody recognizing residues 361–525 cannot detect isoform 2, which lacks that region. For comparisons across samples, establish which isoform or bands the antibody measures and quantify the same bands consistently. Glycosylation at the four listed sites may also complicate assignment of a single band.
Which LAG3 glycosylation sites matter when interpreting bands?
PTM · The listed N-linked sites are Asn188, Asn250, Asn256, and Asn343 in UniProt coordinates. Glycosylation may contribute to band heterogeneity, but the site annotations alone do not establish a visible shift. Keep the numbering convention explicit when comparing an antibody datasheet or paper.
Does this guide establish induction of LAG3?
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 LAG3?
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 A02869 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 LAG3 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 LAG3 migrate differently from its predicted 57.4 kDa?
Interpretation · The 57.4 kDa prediction is a sequence-based reference. LAG3 has a signal peptide at residues 1–22 and four listed N-linked glycosylation sites. These features can affect the protein present in a sample, but the supplied record gives no observed band, so it cannot establish a shift or its size.
Consider the two listed isoforms, four N-linked glycosylation sites, and LAG3’s homodimer annotation as possibilities to investigate. None proves the identity of an unexpected band. Check its antibody recognition region and compare bands under consistent sample preparation conditions before assigning it.
Canonical LAG3 is annotated as a single-pass type I cell-membrane protein. Use a preparation that retains membrane proteins when assessing canonical LAG3. Isoform 2 lacks residues 361–525, so interpret results from a membrane-only preparation with its sequence difference in mind.
Consider the two listed isoforms, four N-linked glycosylation sites, and LAG3’s homodimer annotation as possibilities to investigate. None proves the identity of an unexpected band. Check its antibody recognition region and compare bands under consistent sample preparation conditions before assigning it.
Canonical LAG3 is annotated as a single-pass type I cell-membrane protein. Use a preparation that retains membrane proteins when assessing canonical LAG3. Isoform 2 lacks residues 361–525, so interpret results from a membrane-only preparation with its sequence difference in mind.
Boster reagents
LAG3 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-LAG3 Antibody
Cat # A02869
Real WB data
Anti-LAG3 Monoclonal Antibody [2G8]
Cat # M02869-1
Two anti-LAG3 antibodies are listed with WB images: A02869 was shown on mouse liver tissue lysate at 1 μg/mL; M02869-1 was shown on LAG3-overexpressing HEK293 cells at 0.25–1 μg/mL. These images document those specific sample conditions.
Which to pick: For mouse samples, consider A02869, which lists mouse reactivity and has a mouse liver WB image. For human samples, both list human reactivity; M02869-1 has a WB image from overexpressing HEK293 cells, while A02869’s shown sample is mouse liver.
Source: BosterBio LAG3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P18627.
- Human Protein Atlas. LAG3 tissue expression.
- PMC9589981 — target-verified WB comparison
- PMC13401121 — target-verified WB comparison
- PMC10540310 — target-verified WB comparison