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CYP8B1 / 7-alpha-hydroxycholest-4-en-3-one 12-alpha-hydroxylase · Western blot design guide
Design a Western Blot for CYP8B1
Real validated CYP8B1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CYP8B1 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
CYP8B1 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 | ~58.1 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 | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated CYP8B1 Western Blot Protocols
The A05518-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | HEK293T, sp2/0, PC12 (catalog A05518-2) |
| 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 | A05518-2; 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 CYP8B1 Western Blot Band Size?
CYP8B1 is predicted at 58.1 kDa; its documented phosphoserine and membrane location do not demonstrate altered migration, and no empirical band size is supplied.
| Band near 58.1 kDa | Compatible with full-length CYP8B1; identity needs confirmation |
| Band enriched in microsomal fraction | Consistent with its ER and microsome membrane location |
| Weak band in soluble fraction | Consistent with membrane association |
| Doublet near 58.1 kDa | Phosphoserine 326 is documented, but the cause of the doublet is unproven |
💡Expected CYP8B1 appearanceFull-length CYP8B1 has a predicted mass of 58.1 kDa; no empirical band size is supplied, so confirm any candidate band with ordinary identity controls.
| Predicted molecular mass | Full-length CYP8B1 is calculated at 58.1 kDa |
| 501-residue sequence | Defines the full-length protein used for the predicted mass |
| Phosphoserine 326 | Its effect on apparent migration is not established |
| ER and microsome membrane location | May affect recovery by fraction, without establishing a size shift |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated CYP8B1 may be poorly recovered | Check membrane extraction and a microsomal fraction |
| Band higher than expected | The supplied features do not establish a higher migrating form | Check antibody specificity and compare a CYP8B1-depleted control |
| Band lower than expected | The supplied features do not establish a smaller mature form | Check sample integrity and band identity with a CYP8B1-depleted control |
| Multiple bands | Only one isoform is listed; the extra bands are unassigned | Test which bands disappear after CYP8B1 depletion |
| Weak or no signal | Membrane extraction may yield little target | Check recovery in a microsomal fraction and verify assay performance |
Experiment evidence
Western blot images showing CYP8B1 band
A05518-2 · CYP8B1
A05518-1 · CYP8B1
A05518-1 · CYP8B1
A05518-1 · CYP8B1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CYP8B1 Western blot
🧪For positive controls for CYP8B1 in Western blot, you can use a validated positive sample once identified; the supplied HPA evidence names none.
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 the samples.
⚠️Feasibility: CYP8B1 is an ER membrane protein, and the supplied HPA evidence cannot identify suitable tissue controls.
HPA tissue expression evidence for CYP8B1
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 CYP8B1 Western Blot Tips
Deeper troubleshooting and optimisation questions for CYP8B1, answered from its protein features.
Where should the CYP8B1 band appear?
Band shift · The predicted mass is 58.1 kDa, but no observed band position is supplied. Use 58.1 kDa as a reference, not an exact apparent mass; the listed features do not establish a band shift.
Could CYP8B1 isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It therefore provides no documented isoform explanation for multiple bands.
Does phosphorylation affect CYP8B1 band interpretation?
PTM · UniProt lists phosphoserine at position 326, using UniProt sequence numbering. This identifies a modification site but does not establish whether phosphorylation produces a visible shift. Check numbering conventions before comparing it with antibody or paper annotations.
Does this guide establish induction of CYP8B1?
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 CYP8B1 Western blot?
Transfer · CYP8B1 is listed as a 501-residue, single-pass ER and microsome membrane protein with a predicted mass of 58.1 kDa. The supplied features do not specify a transfer method. Verify transfer around the expected mass by checking the gel and membrane.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A05518-2 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 CYP8B1 bands be quantified?
Quantitation · Compare samples prepared consistently, especially when using ER or microsome fractions, where CYP8B1 is located. Quantify the band identified as CYP8B1 against an appropriate loading reference for the same samples; the supplied record gives no observed band position.
How should unexpected CYP8B1 bands be interpreted?
Interpretation · Check band identity before assigning a cause. The record lists one isoform, no glycosylation sites, and no signal or propeptide, so those features do not explain extra bands. Phosphoserine 326 is documented, but its presence alone does not establish a visible shifted band.
Boster reagents
CYP8B1 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-CYP8B1 Antibody
Cat # A05518-2
Real WB data
Anti-CYP8B1 Antibody
Cat # A05518-1
The catalog reports two anti-CYP8B1 polyclonal antibodies with WB images. A05518-2's caption names HEK293T, sp2/0, and PC12 lysates at 1:500 dilution. A05518-1's caption says only 'various cells.' The supplied evidence gives no further validation details.
Which to pick: Choose A05518-2 if you need listed rat reactivity or a WB image with named lysates and dilution; it also lists human and mouse reactivity. A05518-1 lists human and mouse reactivity, but its WB caption does not identify samples or conditions.
Source: BosterBio CYP8B1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9UNU6.
- Human Protein Atlas. CYP8B1 tissue expression.
- PMC4811446 — target-verified WB comparison