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- Table of Contents
NLGN3 / Neuroligin-3 · Western blot design guide
Design a Western Blot for NLGN3
Source-linked NLGN3 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NLGN3 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
NLGN3 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 | ~93.9 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Cerebellum (IHC candidate; verify WB) +2 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Source-Linked NLGN3 Western Blot Protocol Options
The A04627 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | HEK293T, sp2/0 (catalog A04627) |
| Gel % | 8–10% (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 | A04627; use the WB datasheet starting dilution (standard starting point) |
| 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 NLGN3 Western Blot Band Size?
NLGN3 has a predicted full-length mass of 93.9 kDa; signal-peptide cleavage, glycosylation, and isoforms may affect migration, but no empirical band size demonstrates their effects.
| Band near 93.9 kDa | consistent with the predicted full-length NLGN3 mass; confirm identity with appropriate controls |
| Band above 93.9 kDa | may reflect N-linked glycosylation at Asn98 or Asn545; the size effect is unmeasured |
| Band below 93.9 kDa | may reflect removal of the 1–37 signal peptide; its apparent size is unmeasured |
| Band near twice the monomer size | could reflect an NLGN3 homodimer if it remains associated during electrophoresis |
| Several bands at different sizes | could reflect isoforms 1, 2, and 3; distinct migration is unestablished |
💡Expected NLGN3 appearanceThe full-length predicted mass is 93.9 kDa; no empirical band size is supplied, so identify any candidate band with appropriate controls before assigning effects to processing, glycosylation, or isoforms.
| UniProt predicted mass | 93.9 kDa for the full-length sequence; apparent migration is unmeasured |
| N-linked glycosylation at Asn98 | may increase apparent size; a visible shift is unestablished |
| N-linked glycosylation at Asn545 | may increase apparent size; a visible shift is unestablished |
| Signal peptide at residues 1–37 | cleavage makes the mature protein smaller than the precursor; migration is unmeasured |
| NLGN3 homodimer | could appear near twice the monomer size if it remains associated during electrophoresis |
| Isoforms 1, 2, and 3 | may differ in size; their masses and migration differences are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | NLGN3 is a membrane protein and may be poorly recovered in the preparation | check membrane enrichment and verify a candidate band with an NLGN3-specific control |
| Band higher than expected | glycosylation or a homodimer that remains associated may affect migration | compare glycosidase-treated samples and denaturing conditions, then confirm identity |
| Band lower than expected | signal-peptide cleavage may reduce size relative to the full-length precursor | check the antibody epitope and confirm the band with an NLGN3-specific control |
| Broad smear instead of sharp band | heterogeneous N-linked glycosylation is possible but unestablished | compare untreated and glycosidase-treated samples and verify the signal |
| Multiple bands | isoforms 1, 2, and 3 are annotated, but distinct migration is unestablished | use an NLGN3-specific control before assigning bands to isoforms |
| Weak or no signal | membrane-associated NLGN3 may be underrepresented in the sample | check membrane protein recovery and use a suitable positive control |
Experiment evidence
Western blot images showing NLGN3 band
A04627 · NLGN3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NLGN3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NLGN3 in Western blot, you can use cerebellum tissue, where HPA reports high expression.
Positive control: Cerebellum (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: As a synaptic membrane protein, NLGN3 may be difficult to detect in whole-tissue lysate.
HPA tissue expression evidence for NLGN3
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 |
|---|---|---|---|---|
| Cerebellum | molecular layer cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Hippocampus | neuronal cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NLGN3 Western Blot Tips
Deeper troubleshooting and optimisation questions for NLGN3, answered from its protein features.
How should NLGN3 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 NLGN3 isoforms produce different bands?
Isoforms · Three isoforms are listed. Relative to isoform 1, isoform 2 lacks residues 153–172 and isoform 3 lacks residues 153–192. Check whether the antibody epitope is retained in each isoform before assigning bands. These sequence differences do not guarantee resolved bands.
How can glycosylation be assessed on an NLGN3 blot?
PTM · NLGN3 has N-linked glycosylation sites at UniProt Asn98 and Asn545. Compare untreated and appropriately deglycosylated samples on the same blot to test whether glycosylation contributes to migration. A change would need experimental confirmation; the listed sites alone do not predict its size.
UniProt lists phosphoserine at position 745 and phosphotyrosine at position 792. For a phospho-specific antibody, verify that its stated site uses the same numbering convention as UniProt and that its epitope matches. These annotations alone do not establish a detectable band shift.
UniProt lists phosphoserine at position 745 and phosphotyrosine at position 792. For a phospho-specific antibody, verify that its stated site uses the same numbering convention as UniProt and that its epitope matches. These annotations alone do not establish a detectable band shift.
Does this guide establish induction of NLGN3?
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.
What transfer method to use for NLGN3 Western blot?
Transfer · NLGN3 is a single-pass membrane protein with a predicted 93.9 kDa precursor. Check transfer efficiency at the NLGN3 band using a membrane stain and adjust transfer conditions if signal remains in the gel. The supplied features do not identify one preferred transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A04627 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 NLGN3 bands be quantified?
Quantitation · Quantify consistently defined bands across samples and account for the three annotated isoforms and possible glycosylation-dependent migration. Confirm which forms the antibody recognizes before combining band intensities. The supplied features provide no observed band position to use as a fixed boundary.
Why might NLGN3 migrate differently from its predicted 93.9 kDa mass?
Interpretation · The 93.9 kDa prediction is for the precursor. NLGN3 has a signal peptide at residues 1–37 and N-linked glycosylation sites at Asn98 and Asn545. These features may affect the mature protein’s mass or migration, but they do not establish a visible shift. No observed band position is supplied.
NLGN3 is annotated as a homodimer and as a heterodimer with NLGN1 or NLGN2; it also has three disulfide bonds. Compare reducing and nonreducing samples when evaluating a high-mass band. Those features suggest possibilities but do not identify any particular band as a dimer.
NLGN3 is annotated as a homodimer and as a heterodimer with NLGN1 or NLGN2; it also has three disulfide bonds. Compare reducing and nonreducing samples when evaluating a high-mass band. Those features suggest possibilities but do not identify any particular band as a dimer.
Boster reagents
NLGN3 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-Neuroligin-3 NLGN3 Antibody
Cat # A04627
The catalog reports an anti-NLGN3 antibody with stated Human and Mouse reactivity. Its WB image shows HEK293T and sp2/0 whole-cell lysates at 1:500 dilution; evidence beyond these tested samples is not supplied.
Which to pick: A04627 is the only listed NLGN3 antibody. It has a WB image using HEK293T and sp2/0 whole-cell lysates; consider whether those tested samples and the stated Human/Mouse reactivity fit your experiment.
Source: BosterBio NLGN3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9NZ94.
- Human Protein Atlas. NLGN3 tissue expression.
- PMC10123180 — target-verified WB comparison
- PMC5891832 — target-verified WB comparison
- PMC8415229 — target-verified WB comparison