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- Table of Contents
SLC25A5 / ADP/ATP translocase 2 · Western blot design guide
Design a Western Blot for SLC25A5
Source-linked SLC25A5 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SLC25A5 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
SLC25A5 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 | ~32.9 kDa | |
| Observed band | ~33 and 60 kDa | |
| Gel | 10% (catalog A06393-1) | |
| Positive control | Cerebellum (IHC candidate; verify WB) +4 more | |
| Negative control | Epididymis (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | ANT1/2 cross-reactivity | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked SLC25A5 Western Blot Protocol Options
The A06393-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human K562, human HepG2, rat brain, rat liver, mouse brain, mouse liver, mouse heart (catalog A06393-1) |
| Gel % | 10% (catalog A06393-1) |
| Load | 30 ug; reducing conditions (catalog A06393-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A06393-1) |
| Membrane | nitrocellulose membrane (catalog A06393-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A06393-1) |
| Primary antibody | A06393-1 · 1:1000 (catalog A06393-1) |
| Primary incubation | overnight at 4°C (catalog A06393-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A06393-1) |
| Secondary incubation | 1.5 hour at RT (catalog A06393-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A06393-1) |
| Detection | ECL (catalog A06393-1) |
Section 2
What Is the Expected SLC25A5 Western Blot Band Size?
SLC25A5 is predicted at 32.9 kDa; antibody QC reports approximately 33 and 60 kDa, and the cause of the 60 kDa signal is not established.
| Band near 33 kDa | Matches the 32.9 kDa predicted monomer and the reported antibody signal |
| Band near 60 kDa | Reported antibody signal whose identity is not established by the supplied features |
| Bands near 33 and 60 kDa | Both signals were reported, but the 60 kDa signal requires an identity control |
| Weak or absent band in whole-cell lysate | The inner-membrane protein may be poorly recovered during extraction |
💡Expected SLC25A5 appearanceThe predicted monomer is 32.9 kDa; antibody QC reports bands near 33 and 60 kDa, so use band-identity controls, particularly for the unexplained 60 kDa signal.
| Predicted monomer mass | Sets a 32.9 kDa reference, consistent with the reported 33 kDa band |
| N-terminal processing at residue 2 | Is annotated, but no distinct band size is established |
| Lysine 52 methylation | Is annotated in alternate forms, but no visible size difference is established |
| Serine 7 phosphorylation | Is annotated, but no visible size difference is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Inner-membrane protein may be poorly extracted | Check extraction using a mitochondrial membrane fraction |
| Band higher than expected | The reported 60 kDa signal has no established explanation in the supplied features | Confirm its identity with an independent antibody or SLC25A5 depletion |
| Band lower than expected | Identity of a smaller signal is unestablished | Compare with the 33 kDa reference and verify specificity by SLC25A5 depletion |
| Multiple bands | Antibody QC reports 33 and 60 kDa signals without establishing both identities | Verify each band with an independent antibody or SLC25A5 depletion |
| Weak or no signal | Recovery of the inner-membrane protein may be low | Assess membrane extraction and sample loading |
Experiment evidence
Western blot images showing SLC25A5 band
A06393-1 · SLC25A5
A06393 · SLC25A5
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SLC25A5 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for SLC25A5 in Western blot, you can use cerebellum tissue, which HPA scores as High.
Positive control: Cerebellum (IHC candidate; verify WB)
Negative control: Epididymis (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: Its multi-pass membrane localization may require efficient solubilization for a clear signal.
HPA tissue expression evidence for SLC25A5
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 |
|---|---|---|---|---|
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | High | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | High | Protein (IHC) | HPA → |
| Rectum | glandular cells | High | Protein (IHC) | HPA → |
| Stomach | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Epididymis | glandular cells | Not detected | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Vagina | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced SLC25A5 Western Blot Tips
Deeper troubleshooting and optimisation questions for SLC25A5, answered from its protein features.
How should SLC25A5 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 an isoform explain a second band?
Isoforms · Only one isoform is supplied, with no alternative sequence. The listed features therefore provide no isoform-based explanation for a second band.
Which modifications matter when comparing bands?
PTM · UniProt lists N-terminal acetylation or processing at positions 1 and 2, phosphorylation at 7, and lysine modifications at 23, 43, 52, 92, 96, 105, 147, 163, 166, and 268. Positions 52, 105, 147, and 268 have alternate modifications. These are UniProt sequence coordinates; antibody or paper numbering may differ. Their presence alone does not establish a visible band shift.
Does this guide establish induction of SLC25A5?
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 SLC25A5 Western blot?
Transfer · SLC25A5 is a 32.9 kDa multi-pass membrane protein. Check both the post-transfer gel and membrane to assess transfer near 33 kDa and, if relevant, 60 kDa. The supplied features do not establish one optimal transfer method.
Should blocking conditions be optimized for SLC25A5?
Blocking · SLC25A5 is a multi-pass membrane protein. Compare blockers using the same sample and antibody dilution, then choose the condition with the clearest specific band and lowest background. The supplied features do not specify a preferred blocker.
How should SLC25A5 bands be quantified?
Quantitation · Quantify the ~33 kDa band separately from any ~60 kDa band. Confirm signal linearity and band identity before combining measurements; the UniProt features do not establish that both bands represent SLC25A5.
How should the ~33 and ~60 kDa bands be interpreted?
Interpretation · The predicted mass is 32.9 kDa, consistent with the reported ~33 kDa band. The listed features do not establish the identity of the ~60 kDa band or explain its mobility. Verify each band with an independent antibody or SLC25A5 depletion before assigning it.
SLC25A5 is described as a monomer by similarity and as a component of the MMXD complex. Neither annotation demonstrates that an SDS-resistant complex produces a ~60 kDa band. Treat that band as unassigned until experimentally verified.
SLC25A5 is described as a monomer by similarity and as a component of the MMXD complex. Neither annotation demonstrates that an SDS-resistant complex produces a ~60 kDa band. Treat that band as unassigned until experimentally verified.
Boster reagents
SLC25A5 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-ANT1/2 Antibody
Cat # A06393-1
Real WB data
Anti-ADT2 SLC25A5 Antibody
Cat # A06393
Both listed antibodies report Human, Mouse, and Rat reactivity and include WB images. A06393-1 shows human cell and rodent tissue lysates, with bands near 33 and 60 kDa despite a 33 kDa expected size. A06393 shows SH-SY5Y lysates only.
Which to pick: Choose A06393-1 for a closer match to its documented human cell or mouse and rat tissue samples. A06393 has a WB image from SH-SY5Y cells. Both list the same species reactivity; the images establish only their reported sample contexts.
Source: BosterBio SLC25A5 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P05141.
- Human Protein Atlas. SLC25A5 tissue expression.
- PMC8903613 — target-verified WB comparison
- PMC13207500 — target-verified WB comparison
- PMC13167451 — target-verified WB comparison