This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
NPB / Neuropeptide B · Western blot design guide
Design a Western Blot for NPB
Source-linked NPB Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NPB 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
NPB 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 | ~13.1 kDa | |
| Gel | 15% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Cleaved | |
| Caveat | Precursor processing | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked NPB Western Blot Protocol Options
The A08221 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 A08221) |
| Gel % | 15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.2 µm PVDF (standard starting point) |
| Blocking | Blocking buffer: 3% nonfat dry milk in TBST (catalog A08221) |
| Primary antibody | A08221 · 1:1000 (catalog A08221) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Goat Anti-Rabbit IgG, 1:10000 (catalog A08221) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL (catalog A08221) |
Section 2
What Is the Expected NPB Western Blot Band Size?
NPB has a predicted 13.1 kDa precursor; processing may yield smaller forms, but no migrated band size is demonstrated.
| Band near 13.1 kDa | consistent with the predicted NPB precursor, pending identity controls |
| Band below 13.1 kDa | could reflect signal peptide cleavage |
| Smaller band below the precursor | could reflect propeptide processing; its migration is unknown |
| Little or no band in whole-cell lysate | consistent with secretion of NPB |
💡Expected NPB appearanceUniProt predicts a 13.1 kDa precursor; signal peptide and propeptide processing may produce smaller species, but no empirical band size is supplied, so confirm band identity with antibody and secretion controls.
| 13.1 kDa predicted precursor mass | provides the reference size for unprocessed NPB |
| Signal peptide at residues 1–24 | cleavage may yield a smaller form than the precursor |
| Propeptide at residues 56–125 | processing may yield a smaller peptide of unspecified apparent size |
| Cleavage on pair of basic residues | supports precursor processing but does not establish a band size |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | NPB is secreted | check conditioned medium alongside lysate |
| Band lower than expected | signal peptide or propeptide processing may reduce size | check antibody epitope coverage and compare precursor with secreted material |
| Multiple bands | precursor and processed NPB forms may coexist | compare lysate and conditioned medium and verify bands with an independent antibody |
| Weak or no signal | secretion may leave little NPB in the sampled lysate | test conditioned medium and include a positive control |
| Fragments below expected size | propeptide processing may produce smaller NPB species | check antibody epitope coverage and compare with a fresh sample |
Experiment evidence
Western blot images showing NPB band
A08221 · NPB
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NPB Western blot
🧪For positive controls for NPB in Western blot, you can use no HPA-supported positive sample because tissue expression data are unavailable.
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.
⚠️Feasibility: NPB is secreted, so conditioned medium may be more suitable than whole-cell lysate.
HPA tissue expression evidence for NPB
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 NPB Western Blot Tips
Deeper troubleshooting and optimisation questions for NPB, answered from its protein features.
How should NPB 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 NPB isoforms explain multiple bands?
Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. It therefore provides no isoform-based explanation for multiple bands. Consider the annotated processing regions and whether the antibody recognizes each detected band.
Do listed modifications explain an NPB band shift?
PTM · No glycosylation sites or modified residues are listed in the supplied record. Do not assign a shifted band to a specific modification from these features. Processing is annotated, but its presence alone does not establish a visible shift or explain an apparent mass difference.
Does this guide establish induction of NPB?
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 NPB?
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 A08221 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.
What matters when quantifying secreted NPB?
Quantitation · UniProt places NPB in the secreted compartment and annotates precursor processing. State whether the blot measures a secreted sample or a cell-associated sample, and use an antibody whose target region matches the NPB form being quantified. The record does not specify which form predominates in either sample.
Should NPB run at its predicted 13.1 kDa?
Interpretation · 13.1 kDa is the predicted mass of the 125-residue precursor. UniProt lists a signal peptide at residues 1..24 and a propeptide at 56..125, so processing may change which NPB fragment an antibody detects. No observed band size is supplied; these features alone do not establish a visible shift.
UniProt identifies NPB as secreted and notes cleavage on pairs of basic residues. It also annotates the signal peptide at 1..24 and propeptide at 56..125. Interpret a band in light of the antibody's target region; the supplied features do not establish which processed forms appear on the blot.
Check the antibody epitope against UniProt's signal peptide (1..24) and propeptide (56..125). An antibody targeting a region removed during processing may miss another NPB form. These coordinates use the supplied UniProt precursor numbering.
Compare each band with the 13.1-kDa precursor prediction and the antibody's target region. The signal peptide (1..24), propeptide (56..125), and basic-residue cleavage annotation make processing relevant, but none proves the identity of an unexpected band. An observed NPB band size was not supplied.
UniProt identifies NPB as secreted and notes cleavage on pairs of basic residues. It also annotates the signal peptide at 1..24 and propeptide at 56..125. Interpret a band in light of the antibody's target region; the supplied features do not establish which processed forms appear on the blot.
Check the antibody epitope against UniProt's signal peptide (1..24) and propeptide (56..125). An antibody targeting a region removed during processing may miss another NPB form. These coordinates use the supplied UniProt precursor numbering.
Compare each band with the 13.1-kDa precursor prediction and the antibody's target region. The signal peptide (1..24), propeptide (56..125), and basic-residue cleavage annotation make processing relevant, but none proves the identity of an unexpected band. An observed NPB band size was not supplied.
Boster reagents
NPB 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-Neuropeptide B NPB Antibody
Cat # A08221
A08221 is an anti-NPB antibody listed for human and mouse reactivity. Its Western blot image uses cell line extracts at 25 µg per lane and a 1:1000 primary dilution. The caption does not identify the cell lines or establish species-specific performance.
Which to pick: A08221 is the only listed option. It has a Western blot image with reported sample loading and dilution conditions; check whether its listed human and mouse reactivity fits your sample.
Source: BosterBio NPB gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q8NG41.
- Human Protein Atlas. NPB tissue expression.
- PMC10094185 — target-verified WB comparison
- PMC7659951 — target-verified WB comparison