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- Table of Contents
CYP2C9 / Cytochrome P450 2C9 · Western blot design guide
Design a Western Blot for CYP2C9
Real validated CYP2C9 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CYP2C9 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
CYP2C9 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 | ~55.6 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Liver (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 | 2 isoform(s) |
Section 1
Real Curated CYP2C9 Western Blot Protocols
The M00465-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | HepG2 cell lysate (catalog M00465-1) |
| 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 | M00465-1; 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 CYP2C9 Western Blot Band Size?
CYP2C9 is predicted at 55.6 kDa; two isoforms could affect migration, but no distinct band sizes or empirical migration are demonstrated.
| Band near 55.6 kDa | consistent with the predicted CYP2C9 polypeptide size; verify band identity |
| Two bands of different sizes | isoforms 1 and 2 are possible contributors if their migration differs; confirm their identities |
| One band despite two annotated isoforms | the isoforms may not resolve or both may not be detected |
| Weak or absent band in whole-cell lysate | recovery of this endoplasmic reticulum membrane protein may be limited |
💡Expected CYP2C9 appearanceCYP2C9 has a predicted mass of 55.6 kDa, but no empirical band size is supplied; assess a band near that size with appropriate identity controls.
| Predicted molecular mass | sets the reference at 55.6 kDa |
| 490-residue sequence | defines the full-length polypeptide underlying the predicted mass |
| Isoform 1 | has no supplied isoform-specific mass or migration |
| Isoform 2 | could differ in size, but its mass and migration are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | limited recovery of CYP2C9 from the endoplasmic reticulum membrane | check a microsomal fraction and confirm lysate loading |
| Band higher than expected | no supported modification or assembly explains the position | verify specificity with an independent antibody or CYP2C9 depletion |
| Band lower than expected | isoform expression is possible, but the band identity is unestablished | compare isoform-specific expression or confirm identity by depletion |
| Multiple bands | two isoforms are annotated, but distinct migration is unconfirmed | use isoform-specific controls or CYP2C9 depletion to identify the bands |
| Weak or no signal | low recovery of the membrane-associated protein | check microsomal enrichment and sample loading |
Experiment evidence
Western blot images showing CYP2C9 band
M00465-1 · CYP2C9
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CYP2C9 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CYP2C9 in Western blot, you can use liver tissue lysate.
Positive control: Liver (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside.
⚠️Feasibility: CYP2C9 is membrane-associated, so a microsomal fraction may improve detection.
HPA tissue expression evidence for CYP2C9
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 |
|---|---|---|---|---|
| Liver | hepatocytes | High | Protein (IHC) | HPA → |
| Duodenum | glandular 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | endocrine cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CYP2C9 Western Blot Tips
Deeper troubleshooting and optimisation questions for CYP2C9, answered from its protein features.
Where should the canonical CYP2C9 band appear?
Band shift · The 490-residue canonical sequence has a predicted mass of 55.6 kDa. Use that as a reference, not an exact migration position; no observed band size is supplied.
Could CYP2C9 isoforms produce different bands?
Isoforms · Yes. Isoform 2 replaces residues 161–162 (AS) with GG and lacks residues 163–490, using the supplied UniProt numbering. It is much shorter than canonical isoform 1, so consider it when assessing a lower band. These features alone do not establish that either band will be visible.
Check its epitope against the supplied sequences. An epitope within canonical residues 163–490 would be absent from isoform 2; one within residues 1–160 is shared. Confirm the antibody’s stated epitope before assigning a band to either isoform.
Check its epitope against the supplied sequences. An epitope within canonical residues 163–490 would be absent from isoform 2; one within residues 1–160 is shared. Confirm the antibody’s stated epitope before assigning a band to either isoform.
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 CYP2C9?
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 CYP2C9 Western blot?
Transfer · The supplied features identify a 55.6 kDa predicted protein associated with ER and microsome membranes, but do not specify a transfer method. Check recovery on the receiving membrane and residual protein in the gel before adjusting transfer conditions.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M00465-1 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 CYP2C9 bands be quantified across samples?
Quantitation · Use the same sample fraction and a consistent band assignment across samples. Because isoform 2 lacks residues 163–490, verify that the antibody detects the species being quantified. The supplied features do not establish that a band represents total CYP2C9.
Which sample fraction is relevant for CYP2C9 detection?
Interpretation · CYP2C9 is annotated at the endoplasmic reticulum and microsome membranes as a peripheral membrane protein. Include a membrane or microsomal fraction when evaluating detection, and keep fraction preparation consistent across samples.
Compare them with the 55.6 kDa canonical prediction and the much shorter isoform 2 sequence. Confirm whether the antibody epitope exists in each isoform. No observed band size or annotated modified residues are supplied, so these features cannot establish the identity or cause of an unexpected band.
Compare them with the 55.6 kDa canonical prediction and the much shorter isoform 2 sequence. Confirm whether the antibody epitope exists in each isoform. No observed band size or annotated modified residues are supplied, so these features cannot establish the identity or cause of an unexpected band.
Boster reagents
CYP2C9 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 2C9 Rabbit Monoclonal Antibody
Cat # M00465-1
The catalog reports one anti-CYP2C9 antibody for Western blot: rabbit monoclonal M00465-1. Its reported WB image shows Cytochrome P450 2C9 expression in HepG2 cell lysate; this example does not establish performance across other specimens.
Which to pick: M00465-1 is the only listed option. It reports human reactivity and has a WB image using HepG2 cell lysate. For other sample types, the supplied evidence does not establish performance.
Source: BosterBio CYP2C9 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P11712.
- Human Protein Atlas. CYP2C9 tissue expression.
- PMC13321531 — target-verified WB comparison
- PMC2410158 — target-verified WB comparison
- PMC7912942 — target-verified WB comparison