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- Table of Contents
MT-CO3 / Cytochrome c oxidase subunit 3 · Western blot design guide
Design a Western Blot for MT-CO3
Source-linked MT-CO3 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-MT-CO3 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
MT-CO3 Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~30 kDa | |
| Observed band | ~37 kDa | |
| Gel | 10% (catalog A06912-1) | |
| Positive control | Skin (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | Membrane protein mobility | |
| Regulation | Not found in UniProt | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked MT-CO3 Western Blot Protocol Options
The A06912-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | rat liver, rat kidney, mouse liver, mouse kidney (catalog A06912-1) |
| Gel % | 10% (catalog A06912-1) |
| Load | 30 ug; reducing conditions (catalog A06912-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A06912-1) |
| Membrane | nitrocellulose membrane (catalog A06912-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A06912-1) |
| Primary antibody | A06912-1 · 1:1000 (catalog A06912-1) |
| Primary incubation | overnight at 4°C (catalog A06912-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A06912-1) |
| Secondary incubation | 1.5 hour at RT (catalog A06912-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A06912-1) |
| Detection | ECL (catalog A06912-1) |
Section 2
What Is the Expected MT-CO3 Western Blot Band Size?
MT-CO3 is predicted at 30 kDa, while antibody QC reports about 37 kDa; the cause of the difference is not established.
| Band near 37 kDa | Matches the empirical MT-CO3 band reported in reducing tissue lysates |
| Band near 30 kDa | Runs near the predicted mass; confirm its identity |
| Weak band in whole-cell lysate | Inner-membrane MT-CO3 may be poorly recovered |
| Little band in soluble fraction | MT-CO3 is a mitochondrial inner-membrane protein |
💡Expected MT-CO3 appearanceMT-CO3 has a predicted mass of 30 kDa, while antibody QC reports a band near 37 kDa; the cause is unestablished, so confirm band identity with appropriate controls.
| UniProt predicted mass of 30 kDa | Provides the sequence-based reference for the MT-CO3 band |
| Calculated mass of 29,951 Da | Corresponds to approximately 30 kDa before considering apparent migration |
| Predicted monomer mass | Does not establish where the protein will migrate on a blot |
| 30 kDa sequence-based estimate | Is lower than the reported apparent band at 37 kDa for an unestablished reason |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Inner-membrane MT-CO3 may be poorly extracted | Check membrane solubilization and test an enriched mitochondrial fraction |
| Band higher than expected | The reported 37 kDa band exceeds the 30 kDa prediction for an unestablished reason | Verify identity with knockdown or an independent antibody |
| Band lower than expected | Possible degradation during sample preparation | Prepare samples cold with protease inhibitors and verify band identity |
| Multiple bands | The identities of additional bands are unresolved | Compare with knockdown or an independent antibody |
| Weak or no signal | Limited recovery of the multi-pass inner-membrane protein | Check extraction efficiency and load an enriched mitochondrial fraction |
| Fragments below expected size | Possible proteolysis during sample preparation | Use protease inhibitors and confirm fragments with an independent antibody |
Experiment evidence
Western blot images showing MT-CO3 band
A06912-1 · MT-CO3
A06912 · MT-CO3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for MT-CO3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for MT-CO3 in Western blot, you can use skin tissue, which has high HPA expression.
Positive control: Skin (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: As a multi-pass mitochondrial inner-membrane protein, MT-CO3 may require effective membrane-protein extraction for a clear signal.
HPA tissue expression evidence for MT-CO3
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 |
|---|---|---|---|---|
| Skin | keratinocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial 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 → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Not detected | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MT-CO3 Western Blot Tips
Deeper troubleshooting and optimisation questions for MT-CO3, answered from its protein features.
How should MT-CO3 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.
Are multiple MT-CO3 isoforms expected?
Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. It provides no isoform-based explanation for multiple bands.
Could an annotated modification explain an MT-CO3 band shift?
PTM · No modified residues or glycosylation sites are listed in the supplied features. These annotations do not support assigning a band shift to a specific modification.
Does this guide establish induction of MT-CO3?
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 MT-CO3 Western blot?
Transfer · MT-CO3 is a 261-residue, multi-pass mitochondrial inner-membrane protein with a predicted mass of 30 kDa. Check transfer efficiency around both 30 and 37 kDa. The supplied features do not establish whether wet or semi-dry transfer works better.
Should blocking conditions be optimized for MT-CO3?
Blocking · MT-CO3 is a multi-pass membrane protein, so assess background and the putative MT-CO3 signal when comparing blocking conditions. The supplied features do not specify a preferred blocker or establish that blocking explains an unexpected band.
How should MT-CO3 bands be quantified?
Quantitation · Quantify a consistently identified MT-CO3 band across samples using the same analysis settings and a suitable loading reference. MT-CO3 is a component of the 14-subunit complex IV; band intensity alone does not measure complex IV activity.
Why might MT-CO3 appear near 37 kDa instead of 30 kDa?
Interpretation · The supplied apparent band is ~37 kDa, while the predicted mass is 30 kDa. MT-CO3 is a multi-pass inner-membrane protein, but its listed features do not establish the cause of this difference. Confirm band identity before attributing the shift to a specific mechanism.
The listed features support one isoform and identify MT-CO3 as a multi-pass inner-membrane protein. They do not assign an identity to additional bands. Verify band specificity before labeling an extra band as an isoform, modification, or complex IV species.
The listed features support one isoform and identify MT-CO3 as a multi-pass inner-membrane protein. They do not assign an identity to additional bands. Verify band specificity before labeling an extra band as an isoform, modification, or complex IV species.
Boster reagents
MT-CO3 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-MTCO3 antibody
Cat # A06912-1
Real WB data
Anti-COX3 MT-CO3 Antibody
Cat # A06912
Two the supplier anti-MT-CO3 antibodies list human, mouse, and rat reactivity. WB captions show A06912-1 with rat and mouse liver and kidney lysates, and A06912 with U2OS lysates. A06912-1 reports a ~37 kDa band versus an expected 30 kDa.
Which to pick: Choose by the documented sample context: A06912-1 has a WB image using rat and mouse tissues; A06912 has one using U2OS cells. Both list the same species reactivity. Consider the reported band-size discrepancy for A06912-1.
Source: BosterBio MT-CO3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P00414.
- Human Protein Atlas. MT-CO3 tissue expression.
- PMC7546765 — target-verified WB comparison
- PMC12231110 — target-verified WB comparison