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- Table of Contents
CA9 / Carbonic anhydrase 9 · Western blot design guide
Design a Western Blot for CA9
Real validated CA9 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CA9 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
CA9 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 | ~49.7 kDa | |
| Observed band | ~54 kDa | |
| Gel | 5–20% (catalog A01083-3) | |
| Positive control | Gallbladder (IHC candidate; verify WB) +3 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated CA9 Western Blot Protocols
The A01083-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human U-87MG, human 293T, human Hela (catalog A01083-3) |
| Gel % | 5–20% (catalog A01083-3) |
| Load | 30 ug; reducing conditions (catalog A01083-3) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A01083-3) |
| Membrane | nitrocellulose membrane (catalog A01083-3) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A01083-3) |
| Primary antibody | A01083-3 · 0.5 μg/mL (catalog A01083-3) |
| Primary incubation | overnight at 4°C (catalog A01083-3) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A01083-3) |
| Secondary incubation | 1.5 hour at RT (catalog A01083-3) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A01083-3) |
| Detection | ECL (catalog A01083-3) |
Section 2
What Is the Expected CA9 Western Blot Band Size?
CA9 has a 49.7 kDa predicted precursor and an empirical band near 54 kDa; the cause of that difference is not established.
| Band near 54 kDa | Empirical CA9 band in reducing whole-cell lysate; confirm identity with a CA9-specific control |
| Band below the precursor position | Could reflect removal of the 1–37 signal peptide; identity needs confirmation |
| Higher-mass band under nonreducing conditions | Could reflect disulfide-linked CA9 oligomers |
| Faint or absent lysate band | Membrane-localized CA9 may be poorly recovered during extraction |
💡Expected CA9 appearanceUniProt predicts a 49.7 kDa precursor, while antibody QC shows a band near 54 kDa in reducing whole-cell lysate; the cause of the difference is unestablished, so confirm band identity with controls.
| UniProt precursor mass | Predicts 49.7 kDa; the empirical band is near 54 kDa |
| O-linked GlcNAc at Thr115 | May affect migration; a visible shift is not established |
| N-linked glycosylation at Asn346 | May affect migration; its contribution to the 54 kDa band is unestablished |
| Disulfide-linked oligomers | Can yield higher-mass bands if disulfide links remain intact |
| Signal peptide at residues 1–37 | Cleavage makes the mature chain smaller than the precursor |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-localized CA9 may be poorly extracted | Check membrane protein recovery and run a positive lysate control |
| Band higher than expected | Disulfide-linked oligomers may persist with incomplete reduction | Compare reducing and nonreducing samples |
| Band lower than expected | Signal peptide cleavage may lower the precursor mass | Check band identity with a CA9-specific control |
| Multiple bands | Disulfide-linked oligomers or processed CA9 may contribute | Compare reducing conditions and verify bands with CA9 knockdown |
| Weak or no signal | Membrane extraction or transfer may limit detectable CA9 | Check extraction, transfer, and a positive lysate control |
Experiment evidence
Western blot images showing CA9 band
A01083-3 · CA9
M01083-1 · CA9
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CA9 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CA9 in Western blot, you can use gallbladder tissue, which HPA scores as high expression.
Positive control: Gallbladder (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: CA9 is membrane associated, so membrane-enriched lysate may improve detection.
HPA tissue expression evidence for CA9
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 |
|---|---|---|---|---|
| Gallbladder | glandular cells | High | Protein (IHC) | HPA → |
| Stomach | glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Medium | Protein (IHC) | HPA → |
| Small intestine | 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 | glandular 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 CA9 Western Blot Tips
Deeper troubleshooting and optimisation questions for CA9, answered from its protein features.
How should CA9 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 a second CA9 band represent a listed isoform?
Isoforms · Only one isoform is supplied, with no alternative sequence. Do not assign another band to a CA9 isoform on this evidence alone; verify unexpected bands independently.
Which CA9 glycosylation sites matter when interpreting bands?
PTM · UniProt lists O-linked glycosylation at Thr115 and N-linked glycosylation at Asn346. These are UniProt sequence coordinates; check the numbering convention before comparing them with antibody or paper descriptions. Glycosylation is relevant to band interpretation, but the listed sites do not establish a visible shift.
UniProt lists phosphotyrosine at residue 449, using its sequence numbering. A total-CA9 blot cannot establish phosphorylation at that site. If testing it specifically, use an appropriate phosphotyrosine assay and confirm which numbering convention the reagent uses.
UniProt lists phosphotyrosine at residue 449, using its sequence numbering. A total-CA9 blot cannot establish phosphorylation at that site. If testing it specifically, use an appropriate phosphotyrosine assay and confirm which numbering convention the reagent uses.
Does this guide establish induction of CA9?
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 CA9 Western blot?
Transfer · CA9 is a 459-residue, single-pass membrane protein; its predicted mass is 49.7 kDa and the supplied observed band is approximately 54 kDa. Check transfer recovery around that region on the membrane and post-transfer gel. The supplied features do not identify a preferred transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A01083-3 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 CA9 be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Why might CA9 appear near 54 kDa rather than its predicted 49.7 kDa?
Interpretation · The supplied Western blot observation is approximately 54 kDa, versus 49.7 kDa predicted from the sequence. CA9 has a signal peptide at residues 1–37 and listed glycosylation sites, but those features alone do not establish the cause or size of the difference. Use the observed band as a reference and confirm its identity experimentally.
CA9 is listed at membranes and in the nucleus and nucleolus. Compare equivalent sample preparations and loading across lanes, and quantify the same verified band near the supplied approximately 54 kDa position. Different fractions may contain different amounts of CA9.
UniProt reports disulfide-linked CA9 oligomers. If higher bands appear, compare samples prepared under reducing and nonreducing conditions and verify that the bands contain CA9. An unexpected band cannot be assigned to an oligomer from position alone.
CA9 is listed at membranes and in the nucleus and nucleolus. Compare equivalent sample preparations and loading across lanes, and quantify the same verified band near the supplied approximately 54 kDa position. Different fractions may contain different amounts of CA9.
UniProt reports disulfide-linked CA9 oligomers. If higher bands appear, compare samples prepared under reducing and nonreducing conditions and verify that the bands contain CA9. An unexpected band cannot be assigned to an oligomer from position alone.
Boster reagents
CA9 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-Carbonic Anhydrase 9/CA9 Antibody Picoband®
Cat # A01083-3
Real WB data
Anti-CA9/Ca Ix Rabbit Monoclonal Antibody
Cat # M01083-1
Two the supplier anti-CA9 antibodies have WB images. A01083-3 shows an approximately 54 kDa band in three human cell lysates; M01083-1 shows human stomach lysate. Mouse reactivity is listed for A01083-3, but no mouse WB sample is shown.
Which to pick: For the documented human cell lysate protocol, choose A01083-3; its caption reports samples, conditions, and band size. For human stomach lysate, consider M01083-1, whose WB caption gives fewer details. Only A01083-3 lists mouse reactivity, without a mouse WB example.
Source: BosterBio CA9 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q16790.
- Human Protein Atlas. CA9 tissue expression.
- PMC13440105 — target-verified WB comparison
- PMC7460829 — target-verified WB comparison