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- Table of Contents
SHH / Sonic hedgehog protein · Western blot design guide
Design a Western Blot for SHH
Real validated SHH Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SHH 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
SHH 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 | ~49.6 kDa | |
| Gel | 12–15% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Autocatalytic cleavage products | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated SHH Western Blot Protocols
The PA1072-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Rat Liver, Rat Intestine, HELA (catalog PA1072-1) |
| 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 | PA1072-1; 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 SHH Western Blot Band Size?
UniProt predicts a 49.6 kDa SHH precursor; cleavage and Asn278 glycosylation may affect migration, but no empirical band size or demonstrated shift is supplied.
| Band near 49.6 kDa | Consistent with the predicted full-length SHH precursor; confirm identity with antibody controls |
| Band below 49.6 kDa | May reflect signal peptide removal or autocatalytic processing; fragment size is unspecified |
| Band above 49.6 kDa | Asn278 N-linked glycosylation could contribute, but a visible shift is not established |
| Little or no band in whole-cell lysate | SHH is secreted, so cellular abundance may be low |
💡Expected SHH appearanceUniProt predicts a 49.6 kDa full-length SHH precursor; no empirical band size is supplied, and processing or Asn278 glycosylation cannot establish a specific migration without band-identity controls.
| Full-length precursor mass | UniProt predicts 49.6 kDa before processing |
| Signal peptide at residues 1–23 | Its removal produces a smaller mature protein, with no measured band size supplied |
| N-linked glycosylation at Asn278 | Could increase apparent size, but a visible shift is not established |
| Autocatalytic cleavage | Can yield products below precursor size; product masses are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | SHH is secreted | Check conditioned medium alongside lysate |
| Band higher than expected | Asn278 N-linked glycosylation is possible, but the band identity is unconfirmed | Compare with a deglycosylated sample and verify antibody specificity |
| Band lower than expected | Signal peptide removal or autocatalytic processing may produce smaller species | Check the antibody epitope and compare with a full-length control |
| Multiple bands | Precursor and processed SHH species may coexist | Compare precursor and conditioned-medium samples with an independent SHH antibody |
| Weak or no signal | Secreted SHH may be scarce in the tested sample | Test concentrated conditioned medium with a positive control |
| Fragments below expected size | SHH has an autocatalytic cleavage annotation | Check which processed product the antibody recognizes and confirm with an independent antibody |
Experiment evidence
Western blot images showing SHH band
PA1072-1 · SHH
M00058 · SHH
M00058 · SHH
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SHH Western blot
🧪For positive controls for SHH in Western blot, you can use an HPA-verified positive sample once identified; none is supplied here.
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 such as Ponceau alongside SHH.
⚠️Feasibility: SHH is secreted, so conditioned medium may yield a stronger signal than whole-cell lysate; HPA supplies no tissue controls.
HPA tissue expression evidence for SHH
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 SHH Western Blot Tips
Deeper troubleshooting and optimisation questions for SHH, answered from its protein features.
How should SHH 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.
Should I expect multiple SHH isoforms?
Isoforms · The supplied record lists one isoform and no alternative sequence. Do not assign multiple bands to annotated isoforms on this evidence alone; consider the listed signal peptide and autocatalytic cleavage features when interpreting distinct forms.
Could glycosylation affect an SHH band?
PTM · UniProt lists one N-linked glycosylation site at Asn278, using canonical sequence numbering. Consider this site when assessing a band, but its presence alone does not establish a visible shift. Check whether the band you are measuring contains residue 278.
Does this guide establish induction of SHH?
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 SHH?
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 PA1072-1 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 I quantify SHH when several bands appear?
Quantitation · Define which SHH form and sample compartment you intend to measure, then quantify the corresponding band consistently across samples. The record lists autocatalytic cleavage, an N-product, and both membrane-associated and secreted locations; it does not provide apparent band masses for assigning individual bands.
Why might SHH bands differ from the predicted 49.6 kDa?
Interpretation · The 49.6 kDa prediction is for the 462-residue sequence. SHH has a signal peptide at residues 1–23, and its features indicate autocatalytic cleavage and an N-product. These features suggest distinct protein forms, but they do not establish the apparent mass of any band or explain a particular difference.
The record places SHH at endoplasmic reticulum and Golgi membranes and also describes it as secreted. Choose cell-associated or secreted samples according to the form you intend to measure, and compare like sample types across conditions.
The SHH N-product is annotated as a multimer. If an additional band appears, keep multimerization in mind, but this annotation alone cannot identify that band or predict where it will migrate.
Compare its apparent mass with the 49.6 kDa full-sequence prediction cautiously. The record lists a signal peptide at 1–23, autocatalytic cleavage, an N-product, and N-linked glycosylation at canonical Asn278. None alone confirms the identity or mass of an unexpected band.
The record places SHH at endoplasmic reticulum and Golgi membranes and also describes it as secreted. Choose cell-associated or secreted samples according to the form you intend to measure, and compare like sample types across conditions.
The SHH N-product is annotated as a multimer. If an additional band appears, keep multimerization in mind, but this annotation alone cannot identify that band or predict where it will migrate.
Compare its apparent mass with the 49.6 kDa full-sequence prediction cautiously. The record lists a signal peptide at 1–23, autocatalytic cleavage, an N-product, and N-linked glycosylation at canonical Asn278. None alone confirms the identity or mass of an unexpected band.
Boster reagents
SHH 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-Sonic Hedgehog/SHH Antibody Picoband®
Cat # PA1072-1
Real WB data
Anti-Sonic Hedgehog SHH Rabbit Monoclonal Antibody
Cat # M00058
Both anti-SHH antibodies list human, mouse, and rat reactivity and include WB images. PA1072-1 shows rat tissue and cell lysates; M00058 shows human cell lysates with an approximately 50 kDa band. No publication evidence is supplied.
Which to pick: Choose M00058 for a human whole-cell lysate protocol with reported conditions and a 50 kDa band. Choose PA1072-1 for WB images of rat liver or intestine. Both list mouse reactivity, but neither supplied caption shows a mouse specimen.
Source: BosterBio SHH gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q15465.
- Human Protein Atlas. SHH tissue expression.
- PMC13123099 — target-verified WB comparison
- PMC5749743 — target-verified WB comparison
- PMC5732763 — target-verified WB comparison