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- Table of Contents
PHOX2B / Paired mesoderm homeobox protein 2B · Western blot design guide
Design a Western Blot for PHOX2B
Real validated PHOX2B Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PHOX2B 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
PHOX2B 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 | ~31.6 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Colon (IHC candidate; verify WB) +1 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | — | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated PHOX2B Western Blot Protocols
The A02221 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | 293 cell lysate (catalog A02221) |
| 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 | A02221 · (A) 1 and (B) 2 μg/mL (catalog A02221) |
| 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 PHOX2B Western Blot Band Size?
PHOX2B is predicted at 31.6 kDa; no supplied feature demonstrates altered migration, and no empirical band size is available.
| Single band near 31.6 kDa | Consistent with the predicted PHOX2B mass; confirm identity with controls |
| Band in a nuclear fraction | Consistent with PHOX2B's annotated nuclear location |
| Weak band in whole-cell lysate | Nuclear PHOX2B may be poorly recovered or diluted in the sample |
| Little or no band in a cytoplasmic fraction | Consistent with PHOX2B's annotated nuclear location |
💡Expected PHOX2B appearanceUniProt predicts PHOX2B at 31.6 kDa, but no empirical band size is supplied; confirm any candidate band with identity controls.
| UniProt predicted mass | Places the expected full-length protein near 31.6 kDa |
| Calculated molecular weight | Gives the same predicted mass as 31,621 Da |
| Predicted full-length mass | Provides a reference for assessing candidate bands, without establishing their migration |
| Predicted unmodified mass | Does not establish a shift from 31.6 kDa in the supplied evidence |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear PHOX2B may be poorly recovered | Check nuclear extraction and test a positive-control lysate |
| Weak or no signal | Low PHOX2B abundance or inefficient nuclear protein recovery | Compare nuclear and whole-cell preparations and check antibody performance |
| Band higher than expected | No supplied feature establishes a size-increasing modification | Confirm identity with PHOX2B depletion or an independent antibody |
| Band lower than expected | No supplied feature establishes a cleaved product | Check sample integrity and confirm identity with PHOX2B depletion |
| Multiple bands | The single annotated isoform does not explain additional bands | Use PHOX2B depletion or an independent antibody to identify the specific band |
Experiment evidence
Western blot images showing PHOX2B band
A02221 · PHOX2B
M02221 · PHOX2B
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PHOX2B Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PHOX2B in Western blot, you can use colon tissue, where HPA reports high expression.
Positive control: Colon (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: PHOX2B is nuclear, so a nuclear-enriched lysate may improve detection.
HPA tissue expression evidence for PHOX2B
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 |
|---|---|---|---|---|
| Colon | peripheral nerve/ganglion | High | Protein (IHC) | HPA → |
| Adrenal gland | medullary 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 → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PHOX2B Western Blot Tips
Deeper troubleshooting and optimisation questions for PHOX2B, answered from its protein features.
Where should the main PHOX2B band appear?
Band shift · The supplied UniProt record predicts 31.6 kDa for the 314-residue protein. Use that as a starting point for identifying a band; no empirical apparent mass is supplied, so an exact migration position cannot be established from these features.
Could PHOX2B isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It provides no annotated isoform explanation for multiple bands. Evaluate additional bands against the sample context and antibody specificity.
Do annotated modifications predict a PHOX2B band shift?
PTM · No modified residues or glycosylation sites are listed, and the record lists no signal peptide or propeptide. These features do not establish a modification-driven shift or explain a difference between apparent and predicted mass.
Does this guide establish induction of PHOX2B?
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 PHOX2B?
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 A02221 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 PHOX2B localization guide sample preparation and quantitation?
Quantitation · PHOX2B is annotated as nuclear. Check that the preparation retains nuclear proteins, and compare PHOX2B signals only across samples prepared and loaded consistently. The supplied features do not establish a suitable normalization control.
Could a triplet repeat expansion affect band interpretation?
Interpretation · Triplet repeat expansion is a PHOX2B keyword, and the canonical sequence contains alanine-rich stretches. If the sample carries a documented expansion, consider its sequence when interpreting an unexpected band. The supplied record does not establish the sample genotype or predict an apparent mass for an expanded protein.
UniProt lists an interaction with TRIM11, but that alone does not establish that a higher band is a PHOX2B–TRIM11 complex. Confirm the band's identity before assigning it to this interaction.
UniProt lists an interaction with TRIM11, but that alone does not establish that a higher band is a PHOX2B–TRIM11 complex. Confirm the band's identity before assigning it to this interaction.
Boster reagents
PHOX2B 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-PHOX2B Antibody
Cat # A02221
Real WB data
Anti-PHOX2B Monoclonal Antibody
Cat # M02221
Two anti-PHOX2B antibodies are listed for Western blotting. A02221 has a blot image using 293 cell lysate at 1 and 2 μg/mL; M02221 has one using Neuro-2a cell lysate. These images document those sample contexts, not every listed species.
Which to pick: Choose A02221 for rat samples, since it alone lists rat reactivity. Both list human and mouse reactivity; the shown WB samples are 293 lysate for A02221 and Neuro-2a lysate for M02221. Match your sample to the reported evidence where possible.
Source: BosterBio PHOX2B gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q99453.
- Human Protein Atlas. PHOX2B tissue expression.
- PMC9574099 — target-verified WB comparison
- PMC5641117 — target-verified WB comparison
- PMC13341513 — target-verified WB comparison