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- Table of Contents
NAGA / Alpha-N-acetylgalactosaminidase · Western blot design guide
Design a Western Blot for NAGA
Source-linked NAGA Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NAGA 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
NAGA 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 | ~46.6 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Epididymis (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 + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked NAGA Western Blot Protocol Options
The A04163 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | extracts of various cell lines, (catalog A04163) |
| 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 | Blocking buffer: 3% nonfat dry milk in TBST (catalog A04163) |
| Primary antibody | A04163 · 1:1000 (catalog A04163) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Goat Anti-Rabbit IgG, 1:10000 (catalog A04163) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL (catalog A04163) |
Section 2
What Is the Expected NAGA Western Blot Band Size?
NAGA’s full-length precursor is predicted at 46.6 kDa; cleavage, glycosylation, and homodimerization may affect migration, but no empirical band size is supplied.
| Band near 46.6 kDa | Consistent with the predicted full-length NAGA precursor; identity needs confirmation. |
| Band slightly below 46.6 kDa | Could reflect cleavage of the 1–17 signal peptide; mature migration is not supplied. |
| Band above 46.6 kDa | Could reflect N-linked glycosylation; the migration effect is not established. |
| Band near twice the monomer size under non-reducing conditions | Could reflect a persistent NAGA homodimer. |
💡Expected NAGA appearanceFull-length NAGA is predicted at 46.6 kDa; signal-peptide cleavage, N-linked glycosylation, and homodimerization could affect migration, but no empirical band size is supplied, so confirm band identity with controls.
| Predicted full-length mass | 46.6 kDa is the sequence-based precursor mass, not a measured Western-blot band. |
| N-linked glycosylation at Asn124, Asn177, Asn201, Asn359, and Asn385 | Could increase apparent mass or vary migration; no shift is demonstrated. |
| NAGA homodimer | Could produce a band near twice the monomer size if it persists during electrophoresis. |
| Signal peptide at residues 1–17 | Its cleavage makes the mature protein smaller than the full-length precursor; the apparent size is unknown. |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | N-linked glycosylation or a persistent homodimer is possible. | Compare reducing conditions and assess deglycosylation; confirm identity with NAGA depletion. |
| Band lower than expected | Signal-peptide cleavage could reduce the precursor size. | Check antibody epitope coverage and confirm the band with NAGA depletion. |
| Broad smear instead of sharp band | Heterogeneous N-linked glycosylation is possible but unproven. | Compare untreated and deglycosylated samples, then confirm NAGA-dependent signal. |
| Multiple bands | Precursor and mature forms or differing glycosylation are possible. | Compare deglycosylated samples and identify NAGA-dependent bands by depletion. |
| Weak or no signal | Lysosomal NAGA may be scarce in the sampled whole-cell lysate. | Use a NAGA-positive lysate or enriched lysosomal fraction and verify antibody performance. |
Experiment evidence
Western blot images showing NAGA band
A04163 · NAGA
A04163-1 · NAGA
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NAGA Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NAGA in Western blot, you can use epididymis tissue, which HPA scores High.
Positive control: Epididymis (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: HPA reports adipose tissue as Not detected, providing a tissue negative, though lysosomal abundance may affect signal.
HPA tissue expression evidence for NAGA
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 |
|---|---|---|---|---|
| Epididymis | glandular cells | High | Protein (IHC) | HPA → |
| Pancreas | exocrine glandular cells | High | Protein (IHC) | HPA → |
| Placenta | trophoblastic cells | High | Protein (IHC) | HPA → |
| Seminal vesicle | glandular cells | High | Protein (IHC) | HPA → |
| Skin | fibroblasts | High | 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 → |
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Vagina | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Low | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced NAGA Western Blot Tips
Deeper troubleshooting and optimisation questions for NAGA, answered from its protein features.
How should NAGA 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.
Are multiple NAGA isoforms expected?
Isoforms · The supplied record lists one isoform and no alternative sequence. Do not assign extra bands to splice isoforms on this evidence alone.
Which NAGA glycosylation sites matter when interpreting bands?
PTM · UniProt lists N-linked glycosylation at Asn124, Asn177, Asn201, Asn359, and Asn385. Record the UniProt numbering when comparing with antibody or paper coordinates. These annotations alone cannot assign a particular band to a glycosylated form.
UniProt annotates phosphoserine at residues 322 and 332. Those are UniProt coordinates; check the numbering convention before comparing other sources. The annotations do not show that phosphorylation produces a resolvable Western blot shift.
UniProt annotates phosphoserine at residues 322 and 332. Those are UniProt coordinates; check the numbering convention before comparing other sources. The annotations do not show that phosphorylation produces a resolvable Western blot shift.
Does this guide establish induction of NAGA?
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 NAGA?
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 A04163 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 NAGA bands be quantified across samples?
Quantitation · Use a consistently identified NAGA band across samples and report which band was measured. With no observed band position supplied, the 46.6 kDa prediction alone cannot identify it; avoid combining unexplained bands into one NAGA measurement.
Should NAGA migrate at its predicted 46.6 kDa?
Interpretation · 46.6 kDa is the predicted mass, not an observed Western blot position. NAGA has a signal peptide at residues 1–17 and five annotated N-linked glycosylation sites. These features may affect the protein detected, but the supplied evidence does not establish a visible shift or its size.
NAGA is annotated as a homodimer with four disulfide bonds, and it has five N-linked glycosylation sites. These features make sample preparation and band identity worth checking, but they do not establish that a high band is a dimer or glycosylated NAGA.
No propeptide is annotated. UniProt lists a signal peptide at residues 1–17, but a lower band cannot be assigned to its removal from position alone. Confirm the band's identity before interpreting it as processed NAGA.
NAGA is annotated as a homodimer with four disulfide bonds, and it has five N-linked glycosylation sites. These features make sample preparation and band identity worth checking, but they do not establish that a high band is a dimer or glycosylated NAGA.
No propeptide is annotated. UniProt lists a signal peptide at residues 1–17, but a lower band cannot be assigned to its removal from position alone. Confirm the band's identity before interpreting it as processed NAGA.
Boster reagents
NAGA 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-NAGA Antibody
Cat # A04163
Real WB data
Anti-NAGA Antibody Picoband®
Cat # A04163-1
Both listed anti-NAGA antibodies report human, mouse, and rat reactivity and include WB images. A04163-1 shows a band near 60 kDa, although the caption gives 47 kDa as expected; the supplied evidence does not resolve this difference.
Which to pick: Choose A04163-1 if you want a WB example with named human cell, rat tissue, and mouse tissue lysates. A04163 also has a WB image, but its caption identifies only various cell lines and provides a 1:1000 antibody dilution.
Source: BosterBio NAGA gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P17050.
- Human Protein Atlas. NAGA tissue expression.
- PMC10285022 — target-verified WB comparison