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- Table of Contents
CYP3A5 / Cytochrome P450 3A5 · Western blot design guide
Design a Western Blot for CYP3A5
Real validated CYP3A5 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CYP3A5 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
CYP3A5 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 | ~57.1 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 | — | |
| Caveat | — | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated CYP3A5 Western Blot Protocols
The A00530 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | extracts of various cells, (catalog A00530) |
| 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 | A00530; 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 CYP3A5 Western Blot Band Size?
CYP3A5 is predicted at 57.1 kDa; isoforms could affect migration, but no distinct bands or empirical size are established.
| Band near 57.1 kDa | Consistent with the predicted CYP3A5 size; confirm identity with controls |
| Single sharp band | Compatible with CYP3A5, though annotated isoforms need not resolve separately |
| Several bands at different positions | Isoforms 1 and 2 are annotated, but their migration and band identities are unestablished |
| Faint lysate band with stronger microsomal signal | Consistent with CYP3A5 localization at the microsome and endoplasmic reticulum membrane |
💡Expected CYP3A5 appearanceUniProt predicts CYP3A5 at 57.1 kDa; no empirical band is supplied, and the two annotated isoforms have no established migration difference, so confirm any candidate band with identity controls.
| UniProt predicted molecular weight | Places the reference size near 57.1 kDa |
| Isoform 1 | Its apparent size relative to isoform 2 is unknown |
| Isoform 2 | Its apparent size relative to isoform 1 is unknown |
| Alternative splicing | May affect apparent size, but distinct migration is unestablished |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CYP3A5 is associated with endoplasmic reticulum and microsome membranes | Check a microsomal fraction and a validated positive control |
| Band higher than expected | The annotated isoforms have no supplied migration assignments | Compare with a validated positive control and verify antibody specificity |
| Band lower than expected | The annotated isoforms have no supplied migration assignments | Compare with a validated positive control and verify antibody specificity |
| Multiple bands | Two isoforms are annotated, but their separation is unestablished | Use isoform-aware controls to assign bands |
| Weak or no signal | Membrane localization may limit recovery in the tested fraction | Check microsomal recovery and a validated positive control |
Experiment evidence
Western blot images showing CYP3A5 band
A00530 · CYP3A5
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CYP3A5 Western blot
🧪For positive controls for CYP3A5 in Western blot, you can use an HPA-IHC candidate positive sample once one is identified.
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: HPA provides no tissue candidates, so positive samples need validation; CYP3A5 is membrane associated.
HPA tissue expression evidence for CYP3A5
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 CYP3A5 Western Blot Tips
Deeper troubleshooting and optimisation questions for CYP3A5, answered from its protein features.
How should CYP3A5 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 CYP3A5 isoforms produce different bands?
Isoforms · Two isoforms are listed. In isoform 2, residues 107–140 are replaced and residues 141–502 are missing, using the supplied UniProt coordinates. Its shorter sequence could yield a band distinct from isoform 1, but these features do not establish whether either isoform is expressed or detectable in your sample.
Check the antibody epitope against the supplied isoform sequences. Residues 141–502 of canonical CYP3A5 are missing from isoform 2, while residues 107–140 are replaced. An epitope in either region may prevent recognition of isoform 2. Confirm which numbering convention the antibody supplier uses before comparing residue positions.
Check the antibody epitope against the supplied isoform sequences. Residues 141–502 of canonical CYP3A5 are missing from isoform 2, while residues 107–140 are replaced. An epitope in either region may prevent recognition of isoform 2. Confirm which numbering convention the antibody supplier uses before comparing residue positions.
Do annotated modifications prove a band shift?
PTM · The linked UniProt record describes protein features. A modification annotation alone does not demonstrate a visible shift; retain any condition or experimental qualifier attached to it.
Does this guide establish induction of CYP3A5?
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 CYP3A5 Western blot?
Transfer · The supplied features establish membrane association but do not specify a transfer method. Check transfer efficiency for the candidate band near the canonical predicted mass of 57.1 kDa, and assess sample recovery from the membrane or microsomal fraction before changing transfer conditions.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A00530 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 can I quantify CYP3A5 across samples?
Quantitation · Quantify a consistently identified band within the assay’s linear range. Because isoform 2 lacks canonical residues 141–502, verify that the antibody detects the same isoform or isoforms in every sample. Record which band was measured rather than combining bands without confirming their identities.
Should CYP3A5 run at exactly 57.1 kDa?
Interpretation · 57.1 kDa is the predicted mass of the canonical protein. No observed band position is supplied, so compare candidate bands with a molecular weight marker and confirm their identity experimentally; the predicted mass alone cannot establish where a band will appear.
UniProt places CYP3A5 at the endoplasmic reticulum and microsome membranes as a peripheral membrane protein. Include a membrane or microsomal fraction when assessing recovery, and compare it with your chosen input fraction if a band is weak.
First check whether the antibody epitope is present in isoform 2, which replaces UniProt residues 107–140 and lacks 141–502. Then compare membrane or microsomal enrichment and confirm band identity experimentally. No observed band position, glycosylation sites, or modified residues are supplied; isoform features alone do not explain a particular apparent mass.
UniProt places CYP3A5 at the endoplasmic reticulum and microsome membranes as a peripheral membrane protein. Include a membrane or microsomal fraction when assessing recovery, and compare it with your chosen input fraction if a band is weak.
First check whether the antibody epitope is present in isoform 2, which replaces UniProt residues 107–140 and lacks 141–502. Then compare membrane or microsomal enrichment and confirm band identity experimentally. No observed band position, glycosylation sites, or modified residues are supplied; isoform features alone do not explain a particular apparent mass.
Boster reagents
CYP3A5 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-Cytochrome P450 3A5 CYP3A5 Antibody
Cat # A00530
The catalog reports A00530 as an anti-CYP3A5 antibody for Western blotting, with reported human, mouse, and rat reactivity. Its WB image is described as using extracts from various cells; the supplied evidence does not identify those cells or establish validation in every listed species.
Which to pick: A00530 is the only listed option. It has a WB image, so check the image and reported reactivity against your planned sample before choosing it.
Source: BosterBio CYP3A5 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P20815.
- Human Protein Atlas. CYP3A5 tissue expression.
- PMC10980945 — target-verified WB comparison
- PMC4780258 — target-verified WB comparison
- PMC4018728 — target-verified WB comparison