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LGI1 · Western blot design guide
Design a Western Blot for LGI1
Real validated LGI1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-LGI1 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
LGI1 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 | ~63.8 kDa | |
| Observed band | ~64 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | N-linked glycosylation | |
| Regulation | IL-2-induced | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated LGI1 Western Blot Protocols
Literature-validated Western blot parameters for LGI1 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human SH-SY5Y , Lane 2: rat C6 , Lane 3: mouse Neuro-2a , Lane 4: human HEK293 . After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-Lgi1/EPT/LGI1 antigen affinity purified polyclonal antibody (Catalog # A00850-1) 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 Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for Lgi1/EPT/LGI1 at approximately 64KD. The expected band size for Lgi1/EPT/LGI1 is at 64KD |
| Gel % | 10–12% |
| Load | 50ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 64 kDa |
Section 2
What Is the Expected LGI1 Western Blot Band Size?
LGI1 has a 63.8 kDa precursor backbone but consistently runs at ~64 kDa on blots because N-glycosylation at three sites offsets the mass lost from signal-peptide cleavage.
| single band around 64 kDa | matches mature, glycosylated LGI1 as it is secreted, with signal-peptide loss offset by added N-glycan mass |
| band sitting above the ~60 kDa deglycosylated backbone | reflects added mass from N-linked glycans at Asn192, Asn277, and Asn422 |
| diffuse or smeared band rather than one sharp line | heterogeneous glycan occupancy across the three N-glycosylation sites |
| little or no band in whole-cell lysate | LGI1 is predominantly secreted extracellularly, so lysate can under-represent the mature pool |
| extra closely spaced bands | presence of three annotated splice isoforms (1, 2, 3) differing in length |
| band smaller than the 63.8 kDa full-length precursor | loss of the 34-residue signal peptide during maturation and secretion |
💡Expected LGI1 appearanceExpect a single band at roughly 64 kDa, matching antibody QC data, since N-glycosylation at three sites offsets the mass lost when the 34-residue signal peptide is cleaved from the 63.8 kDa precursor.
| Predicted precursor mass (63.8 kDa) | sets the baseline size before cleavage or modification is considered |
| Signal peptide cleavage (residues 1-34) | removes several kDa from the precursor to generate the mature secreted backbone |
| N-glycosylation at Asn192, Asn277, Asn422 | adds mass back onto the cleaved backbone, pushing the mature protein up toward the ~64 kDa band actually seen on blots |
| Splice isoforms 1, 2, 3 | can generate extra bands of differing size relative to the canonical form, though exact per-isoform mass is not defined |
| Secreted/extracellular localization | shifts much of the detectable protein into secreted fractions, reducing signal from whole-cell lysate alone |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | LGI1 is a secreted protein, so whole-cell lysate may retain little of the mature form | Test conditioned medium or a concentrated secreted fraction alongside lysate |
| Band higher than expected | Full occupancy of the three N-glycosylation sites increases apparent mass above the unmodified backbone | Treat the sample with PNGase F and confirm the band shifts down toward the deglycosylated backbone |
| Band lower than expected | Reduced glycosylation, such as from a non-glycosylating expression system, yields a band closer to the unmodified backbone | Check the expression system of any recombinant standard and note reduced glycosylation as a likely cause |
| Broad smear instead of sharp band | Heterogeneous glycoform occupancy across the three N-glycosylation sites broadens the band | Run a longer resolving gel or treat with PNGase F/Endo H to sharpen the signal |
| Multiple bands | Three annotated splice isoforms may be co-expressed, producing bands of different sizes | Confirm isoform identity via antibody epitope location or transcript-level validation |
| Fragments below expected size | Cleavage of the 34-residue signal peptide during secretion produces a mature form smaller than the annotated precursor | Use fresh lysate with protease inhibitors and confirm whether the antibody targets the pro- or mature-region epitope |
Experiment evidence
Western blot images showing LGI1 band
A00850-1 · LGI1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for LGI1 Western blot
🧪For positive controls for LGI1 in Western blot, you can use conditioned medium or cell lysate from cells transiently transfected to overexpress recombinant LGI1, since no confirmed positive tissue is available from Human Protein Atlas data and native LGI1 is restricted to secreted/synaptic compartments.
Positive control: LGI1-transfected cell lysate/conditioned medium
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin as loading controls alongside a total-protein stain such as stain-free gel, Ponceau S, or REVERT.
⚠️Feasibility: As a secreted protein with no HPA tissue-expression data to anchor a positive/negative pair, whole-cell lysates may show weak native signal, so favor conditioned medium/overexpression systems and confirm specificity with siRNA knockdown or a KO line rather than relying on tissue selection alone.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced LGI1 Western Blot Tips
Deeper troubleshooting and optimisation questions for LGI1, answered from its protein features.
Why does LGI1 run close to its predicted 63.8 kDa mass?
LGI1 has no disulfide bonds and no annotated modified residues, so its core backbone mass is unaltered. Its three N-glycosylation sites add only modest mass, consistent with the observed ~64 kDa band closely matching the 63.8 kDa prediction. A band well above 70 kDa is unlikely from glycosylation alone and more likely reflects a co-migrating complex partner.
Could the three LGI1 isoforms cause extra bands?
LGI1 has three annotated isoforms arising from alternative splicing. Depending on where the antibody epitope falls, one isoform may be undetected while others appear as closely spaced bands near 64 kDa or as a smaller species if a spliced region is missing. Check the immunogen region against isoform sequences before calling extra bands nonspecific.
Does LGI1 glycosylation affect blocking buffer choice?
LGI1 is annotated as a glycoprotein with three N-glycosylation sites. Milk-based blockers contain glycoproteins and lectin-binding sugars that can cross-react with glycosylated targets and raise background. Using BSA-based blocking buffer instead of milk reduces this risk and gives cleaner detection of the glycosylated LGI1 band.
What transfer method to use for LGI1 Western blot?
At 63.8 kDa, LGI1 transfers well with standard wet or semi-dry protocols; no extended transfer time is needed for a protein this size. Because LGI1 is secreted and glycosylated, PVDF membrane is preferred over nitrocellulose for stronger retention of the glycoprotein during blocking and washing steps.
How should secreted LGI1 be normalized for quantitation?
LGI1 is annotated as secreted in addition to cytoplasmic and synaptic pools. If blotting conditioned media or CSF rather than cell lysate, intracellular housekeeping controls like GAPDH are invalid normalizers. Use total protein staining of the membrane or a matched secreted reference protein instead to quantify secreted LGI1 signal.
Why might LGI1 blots show bands above 64 kDa?
LGI1 oligomerizes and forms complexes with KCNA1, KCNA4, KCNAB1, ADAM22, DLG4/PSD95 and CACNG2. Under non-reducing or incompletely denaturing sample prep, these partners or LGI1 oligomers can co-migrate as higher-molecular-weight species above the 63.8 kDa monomer. Use fully reducing, denaturing sample buffer to resolve the true monomeric band.
Do disease-linked LGI1 variants change the band pattern?
LGI1 is annotated with disease variants linked to epilepsy, and its normal path involves ER and Golgi processing before secretion. Mutant forms are often retained intracellularly or misfolded, which can reduce the mature ~64 kDa secreted-fraction signal and introduce additional lower-mobility or smeared species distinct from wild-type LGI1.
Boster reagents
Best LGI1 Western Blot Antibodies
BosterBio's LGI1 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-Lgi1/EPT/LGI1 Antibody Picoband®
Cat # A00850-1
Our top-recommended anti-LGI1 antibody for Western blot has been thoroughly validated, is well cited in the literature, and delivers strong, specific band detection, with performance orthogonally confirmed against negative tissue controls and complementary detection methods for reliable, reproducible results.
Which to pick: Only one Boster antibody is catalogued for LGI1: A00850-1, which includes a genuine Western blot validation image, making it the clear default choice for your LGI1 WB experiments.
Source: BosterBio LGI1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O95970.
- Human Protein Atlas. LGI1 tissue expression.