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SIRT5 / NAD-dependent protein deacylase sirtuin-5, mitochondrial · Western blot design guide
Design a Western Blot for SIRT5
Real validated SIRT5 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SIRT5 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
SIRT5 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 | ~33.9 kDa | |
| Gel | 10% (catalog A02395-3) | |
| Positive control | Adrenal gland (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 | Target-mismatched blot | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 4 isoform(s) |
Section 1
Real Curated SIRT5 Western Blot Protocols
The A02395-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human U251, human SH-SY5Y, human HepG2 (catalog A02395-3) |
| Gel % | 10% (catalog A02395-3) |
| Load | 30 ug; reducing conditions (catalog A02395-3) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A02395-3) |
| Membrane | nitrocellulose membrane (catalog A02395-3) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A02395-3) |
| Primary antibody | A02395-3; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A02395-3) |
| Secondary incubation | 1.5 hour at RT (catalog A02395-3) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A02395-3) |
| Detection | ECL (catalog A02395-3) |
Section 2
What Is the Expected SIRT5 Western Blot Band Size?
SIRT5 has a predicted monomer mass of 33.9 kDa; isoforms and homodimerization could affect bands, but no migration pattern is demonstrated.
| Band near 34 kDa | consistent with the predicted 33.9 kDa SIRT5 monomer; confirm identity with controls |
| Band near 68 kDa | could reflect a homodimer that persists during sample preparation |
| Several bands at different positions | could reflect isoforms 1, 2, 3, and 4; their migration differences are unknown |
| Faint band in whole-cell lysate | may reflect SIRT5 being mainly mitochondrial |
💡Expected SIRT5 appearanceUniProt predicts a 33.9 kDa SIRT5 monomer, but no empirical band size is supplied; confirm a band near that size with antibody specificity controls.
| UniProt predicted monomer mass | sets a 33.9 kDa reference, not a measured band position |
| Monomeric SIRT5 | may migrate near the predicted mass after denaturation |
| SIRT5 homodimer formation | could yield a band near twice the monomer mass if the dimer persists during electrophoresis |
| Splice isoforms 1, 2, 3, and 4 | could differ in apparent size; their masses and separation are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | SIRT5 is mainly mitochondrial, and the lysate signal may be weak | check mitochondrial enrichment and a positive-control sample |
| Band higher than expected | a SIRT5 homodimer may persist during sample preparation | compare denaturing conditions and confirm band identity |
| Band lower than expected | the supplied features do not establish a lower migrating species | check antibody specificity with a SIRT5 depletion control |
| Multiple bands | four splice isoforms are listed, but their band positions are unknown | compare bands with a SIRT5 depletion control |
| Weak or no signal | SIRT5 is mainly mitochondrial | check sample quality and compare a mitochondrial fraction |
Experiment evidence
Western blot images showing SIRT5 band
A02395-3 · SIRT5
M02395 · SIRT5
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SIRT5 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for SIRT5 in Western blot, you can use adrenal gland tissue, which HPA rates High.
Positive control: Adrenal gland (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: A negative tissue is available, but mitochondrial enrichment may improve detection of mainly mitochondrial SIRT5.
HPA tissue expression evidence for SIRT5
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Colon | endothelial cells | High | Protein (IHC) | HPA → |
| Thyroid gland | glandular cells | High | Protein (IHC) | HPA → |
| Urinary bladder | urothelial cells | High | Protein (IHC) | HPA → |
| Appendix | 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 → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | chondrocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Low | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Low | Protein (IHC) | HPA → |
Section 3
Advanced SIRT5 Western Blot Tips
Deeper troubleshooting and optimisation questions for SIRT5, answered from its protein features.
How should SIRT5 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 SIRT5 isoforms produce bands of different sizes?
Isoforms · Yes. Four isoforms are listed. Relative to the canonical sequence, isoform 4 lacks residues 1–108, isoform 3 lacks 189–206, and isoform 2 has a replacement at 286–299 and lacks 300–310. These differences can change calculated mass, but the features do not establish which isoforms yield visible bands.
Check the antibody epitope against the listed sequence changes. An epitope within residues 1–108 may miss isoform 4; one within 189–206 may miss isoform 3. Isoform 2 changes residues 286–299 and lacks 300–310. Coordinates refer to the supplied canonical UniProt sequence.
Check the antibody epitope against the listed sequence changes. An epitope within residues 1–108 may miss isoform 4; one within 189–206 may miss isoform 3. Isoform 2 changes residues 286–299 and lacks 300–310. Coordinates refer to the supplied canonical UniProt sequence.
Does SIRT5 have listed modifications that explain a shifted band?
PTM · No modified residues or glycosylation sites are listed in the supplied features. The features therefore do not identify a modification responsible for a shift. They also do not establish the apparent mass of any SIRT5 band.
Does this guide establish induction of SIRT5?
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.
How should transfer be checked for SIRT5?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02395-3 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 I quantify SIRT5 when multiple bands appear?
Quantitation · Define the band or bands used for quantitation before comparing samples. The four listed isoforms have sequence differences that can affect size and antibody recognition, while no empirical apparent band is supplied. Verify band identity before combining intensities from different bands.
How should I interpret a band near 33.9 kDa?
Interpretation · 33.9 kDa is the predicted mass of the 310-residue canonical SIRT5 sequence. No empirical apparent band is supplied, so compare the band with this reference without treating a match as proof of identity.
SIRT5 is mainly mitochondrial, with locations listed in the matrix and intermembrane space. A fraction is also cytosolic, and very small amounts are detected in the nucleus. Keep the fraction analyzed consistent across samples when comparing band intensity.
SIRT5 is reported as both a monomer and a homodimer, with homodimers forming upon suramin binding. Those interaction features alone do not establish that a higher band on a Western blot is a dimer. Interpret such a band only with supporting evidence.
SIRT5 is mainly mitochondrial, with locations listed in the matrix and intermembrane space. A fraction is also cytosolic, and very small amounts are detected in the nucleus. Keep the fraction analyzed consistent across samples when comparing band intensity.
SIRT5 is reported as both a monomer and a homodimer, with homodimers forming upon suramin binding. Those interaction features alone do not establish that a higher band on a Western blot is a dimer. Interpret such a band only with supporting evidence.
Boster reagents
SIRT5 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-SIRT5 Antibody Picoband®
Cat # A02395-3
Real WB data
Anti-SIRT5/Sirtuin 5 Rabbit Monoclonal Antibody
Cat # M02395
Two anti-SIRT5 antibodies are listed. M02395 has a SIRT5 Western blot caption using HeLa lysate. Although A02395-3 is marked as having a WB image, its supplied caption identifies FRZB and a different antibody, so it does not document SIRT5 performance.
Which to pick: For human HeLa lysate, M02395 has the relevant WB example; mouse reactivity is listed but not shown in its caption. A02395-3 lists human, mouse, and rat reactivity, but its mismatched FRZB caption provides no usable SIRT5 WB example.
Source: BosterBio SIRT5 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9NXA8.
- Human Protein Atlas. SIRT5 tissue expression.
- PMC7058372 — target-verified WB comparison
- PMC13369707 — target-verified WB comparison
- PMC12049830 — target-verified WB comparison