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- Table of Contents
COX5A / Cytochrome c oxidase subunit 5A, mitochondrial · Western blot design guide
Design a Western Blot for COX5A
Real validated COX5A Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-COX5A 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
COX5A 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 | ~16.8 kDa | |
| Observed band | ~15 kDa | |
| Gel | 12% (catalog A07895-2) | |
| Positive control | Appendix (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 | Phosphorylated + Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated COX5A Western Blot Protocols
The A07895-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human 293T, human U2OS (catalog A07895-2) |
| Gel % | 12% (catalog A07895-2) |
| Load | 30 ug; reducing conditions (catalog A07895-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A07895-2) |
| Membrane | nitrocellulose membrane (catalog A07895-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A07895-2) |
| Primary antibody | A07895-2 · 1:1000 (catalog A07895-2) |
| Primary incubation | overnight at 4°C (catalog A07895-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A07895-2) |
| Secondary incubation | 1.5 hour at RT (catalog A07895-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A07895-2) |
| Detection | ECL (catalog A07895-2) |
Section 2
What Is the Expected COX5A Western Blot Band Size?
COX5A is predicted at 16.8 kDa and observed at ~15 kDa; the cause of this difference is not established.
| Band at ~15 kDa | Empirical COX5A band in reducing whole-cell and tissue lysates; confirm identity with a control. |
| Band near 16.8 kDa | Near the UniProt predicted mass; identity still needs confirmation. |
| Weak or absent band in whole-cell lysate | COX5A is associated with the mitochondrial inner membrane and may be underrepresented in the sample. |
| Band above the monomer range | Could reflect incomplete dissociation of the complex IV assembly; identity is unconfirmed. |
💡Expected COX5A appearanceUniProt predicts 16.8 kDa, while antibody QC shows a ~15 kDa band; the cause of the difference is unestablished, so confirm identity with an appropriate control.
| UniProt predicted mass | 16.8 kDa is the sequence-based reference; the empirical band is ~15 kDa. |
| N6-acetyllysine at residue 87 | A documented modification with no demonstrated shift in apparent band size. |
| N6-acetyllysine at residue 113 | A documented modification with no demonstrated shift in apparent band size. |
| Phosphothreonine at residue 141 | A documented modification with no demonstrated shift in apparent band size. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Mitochondrial inner-membrane COX5A may be underrepresented or poorly extracted. | Check mitochondrial recovery and extraction; compare an enriched fraction. |
| Band lower than expected | The observed ~15 kDa band is below the 16.8 kDa prediction; its cause is unestablished. | Confirm band identity with COX5A depletion or another specific control. |
| Band higher than expected | Incomplete dissociation of the complex IV assembly is possible. | Check denaturation and confirm the band with a COX5A-specific control. |
| Multiple bands | Acetylation and phosphorylation are documented, but distinct migrating forms are unproven. | Use COX5A depletion to identify specific bands; assess modification only if warranted. |
| Weak or no signal | COX5A is localized to the mitochondrial inner membrane. | Check sample loading and mitochondrial recovery; assess antibody performance with a positive control. |
Experiment evidence
Western blot images showing COX5A band
A07895-2 · COX5A
A07895-1 · COX5A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for COX5A Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for COX5A in Western blot, you can use appendix tissue, which HPA scores High.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: COX5A is mitochondrial, so whole-cell lysate signal may vary with mitochondrial abundance despite the reported adipose tissue negative.
HPA tissue expression evidence for COX5A
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 |
|---|---|---|---|---|
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuropil | High | Protein (IHC) | HPA → |
| Colon | glandular cells | High | 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 → |
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | fibroblasts | Not detected | Protein (IHC) | HPA → |
| Epididymis | glandular cells | Low | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Low | Protein (IHC) | HPA → |
Section 3
Advanced COX5A Western Blot Tips
Deeper troubleshooting and optimisation questions for COX5A, answered from its protein features.
How should COX5A 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 COX5A isoforms explain multiple bands?
Isoforms · Only one isoform is supplied, with no alternative sequence. The supplied features therefore do not support assigning multiple bands to distinct COX5A isoforms.
Which COX5A modifications matter when interpreting bands?
PTM · UniProt lists N6-acetyllysine at positions 87 and 113 and phosphothreonine at position 141. These are UniProt sequence coordinates; antibody or paper numbering may differ. Their presence alone does not establish a visible band shift.
No glycosylation sites are supplied. The listed modifications are acetylation at UniProt positions 87 and 113 and phosphorylation at position 141. These features alone cannot explain the identity or size of an unexpected band.
No glycosylation sites are supplied. The listed modifications are acetylation at UniProt positions 87 and 113 and phosphorylation at position 141. These features alone cannot explain the identity or size of an unexpected band.
Does this guide establish induction of COX5A?
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 COX5A Western blot?
Transfer · COX5A has an apparent band near 15 kDa, so check retention of low-mass proteins during transfer. The supplied features identify it as a peripheral protein on the matrix side of the mitochondrial inner membrane, but do not establish one required transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A07895-2 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 COX5A bands be quantified?
Quantitation · Measure the band near the supplied ~15 kDa apparent position using exposures within the assay’s linear range. COX5A localizes to mitochondria, so account for mitochondrial content when comparing samples. The features do not establish that every nearby band represents COX5A.
Why might COX5A appear near 15 kDa instead of 16.8 kDa?
Interpretation · The supplied apparent band is ~15 kDa, compared with a predicted mass of 16.8 kDa. A transit peptide is listed as a keyword, but no cleavage position is supplied, so its contribution to the difference cannot be calculated. The listed modifications do not establish a visible shift.
COX5A is part of the 14-subunit cytochrome c oxidase complex, but complex membership alone does not identify a higher band. Compare the unexpected band with the ~15 kDa apparent COX5A band and verify its identity before assigning it to a complex or modification.
COX5A is part of the 14-subunit cytochrome c oxidase complex, but complex membership alone does not identify a higher band. Compare the unexpected band with the ~15 kDa apparent COX5A band and verify its identity before assigning it to a complex or modification.
Boster reagents
COX5A 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-COX5A Antibody
Cat # A07895-2
Real WB data
Anti-COX5A Antibody
Cat # A07895-1
Both listed anti-COX5A antibodies report human, mouse, and rat reactivity and have WB images. A07895-2's caption reports a band near 15 kDa, versus 17 kDa expected. A07895-1's caption names cell lysates and dilution but gives no band size.
Which to pick: Choose A07895-2 for a documented example using human cell or rat and mouse tissue lysates at 1:1000. A07895-1 shows MCF-7, sp2/0, and PC12 lysates at 1:500. Match the reported samples and conditions to your experiment.
Source: BosterBio COX5A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P20674.
- Human Protein Atlas. COX5A tissue expression.
- PMC10299295 — target-verified WB comparison
- PMC7365906 — target-verified WB comparison
- PMC8492744 — target-verified WB comparison