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- Table of Contents
ATG5 / Autophagy protein 5 · Western blot design guide
Design a Western Blot for ATG5
Real validated ATG5 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ATG5 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
ATG5 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 | ~32.4 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Bone marrow (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Acetylated + Ubl conjugation | |
| Caveat | ATG12 conjugate controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated ATG5 Western Blot Protocols
The A00240 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | rat spleen tissue lysate (catalog A00240) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A00240 · (A) 1 and (B) 2 μg/mL (catalog A00240) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected ATG5 Western Blot Band Size?
ATG5 is predicted at 32.4 kDa; ATG12 conjugation and Long and Short isoforms may affect bands, but their migration is unverified.
| Band near 32.4 kDa | Consistent with predicted ATG5 size; confirm identity with a control. |
| Higher band | May represent the ATG12–ATG5 conjugate; its migration is not supplied. |
| Additional band of a different size | Could reflect Long and Short isoforms; distinct migration is not established. |
| Little or no band in lysate | ATG5 may be enriched in the membrane-associated fraction. |
💡Expected ATG5 appearanceATG5 has a predicted mass of 32.4 kDa; its ATG12 conjugate and Long and Short isoforms could affect the pattern, but no empirical migration is supplied, so verify band identity with controls.
| Predicted ATG5 mass | 32.4 kDa is the sequence-based reference, not a validated band position. |
| ATG12–ATG5 conjugation | May yield a higher apparent band; its migration is not supplied. |
| Long and Short isoforms | May differ in size, but their masses and separation are not supplied. |
| N-acetylmethionine at residue 1 | A listed modification with no demonstrated resolvable shift. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | ATG5 may be enriched in a membrane-associated fraction or below detection. | Check fraction recovery, loading, and a positive control. |
| Band higher than expected | ATG12–ATG5 conjugation is possible. | Probe for ATG12 and compare with an ATG5-depleted control. |
| Band lower than expected | The Short isoform is possible, but its migration is unknown. | Confirm identity with an ATG5-depleted control and an isoform-aware reagent. |
| Multiple bands | ATG12 conjugation or Long and Short isoforms may contribute. | Compare ATG5 depletion with ATG12 probing; check antibody specificity. |
| Weak or no signal | Low ATG5 abundance or inadequate recovery is possible. | Check sample loading, fraction recovery, and antibody performance with a positive control. |
Experiment evidence
Western blot images showing ATG5 band
A00240 · ATG5
M00240 · ATG5
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ATG5 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ATG5 in Western blot, you can use appendix tissue lysate, which HPA rates High.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Bone marrow (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside.
⚠️Feasibility: HPA reports a Not detected tissue, though ATG5 signal in bone marrow lysate should be verified.
HPA tissue expression evidence for ATG5
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 → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Hippocampus | glial cells | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ATG5 Western Blot Tips
Deeper troubleshooting and optimisation questions for ATG5, answered from its protein features.
How should ATG5 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 the Long and Short isoforms produce different ATG5 bands?
Isoforms · UniProt supplies the 275-residue canonical ATG5 sequence with a predicted mass of 32.4 kDa. Its VAR_SEQ annotation replaces residues 1–79 with M in the Short isoform. This sequence difference does not establish apparent WB masses or whether the isoforms form resolvable bands. Check which isoform your antibody recognizes.
Does ATG5 acetylation explain a band shift?
PTM · UniProt lists N-acetylmethionine at position 1, using UniProt numbering. This feature alone does not establish a visible shift or explain any difference from the predicted 32.4 kDa mass.
Does this guide establish induction of ATG5?
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 ATG5?
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 A00240 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.
Which ATG5 band should be quantified?
Quantitation · Define whether the measurement concerns ATG5 near its predicted 32.4 kDa mass or ATG12-conjugated ATG5. Quantify consistently identified bands separately; the supplied features do not establish an observed band position or justify combining them.
Why might ATG5 migrate above its predicted 32.4 kDa?
Interpretation · ATG5 forms a conjugate with ATG12, so a higher band could be a candidate for that conjugate. The supplied features do not establish its apparent mass, and no observed band position is available. Confirm band identity before assigning it.
ATG5 forms a conjugate with ATG12 and participates in an ATG12–ATG5–ATG16L1 complex. Consider the conjugate when interpreting a higher band, but do not assign a band from size alone. The reported 400 kDa complex size is not an expected ATG5 band position.
ATG5 is reported in the cytoplasm and at the preautophagosomal structure membrane as a peripheral membrane protein. Its ATG12 conjugate can detach from the membrane around completion of autophagosome formation. Keep sample fractions and preparation consistent when comparing bands.
Consider the listed Long and Short isoforms and ATG12 conjugation as candidates. N-terminal acetylation at UniProt position 1 is also listed, but its presence alone does not demonstrate a visible shift. Verify band identity experimentally rather than assigning it from apparent mass alone.
ATG5 forms a conjugate with ATG12 and participates in an ATG12–ATG5–ATG16L1 complex. Consider the conjugate when interpreting a higher band, but do not assign a band from size alone. The reported 400 kDa complex size is not an expected ATG5 band position.
ATG5 is reported in the cytoplasm and at the preautophagosomal structure membrane as a peripheral membrane protein. Its ATG12 conjugate can detach from the membrane around completion of autophagosome formation. Keep sample fractions and preparation consistent when comparing bands.
Consider the listed Long and Short isoforms and ATG12 conjugation as candidates. N-terminal acetylation at UniProt position 1 is also listed, but its presence alone does not demonstrate a visible shift. Verify band identity experimentally rather than assigning it from apparent mass alone.
Boster reagents
ATG5 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-Autophagy protein 5 ATG5 Antibody
Cat # A00240
Real WB data
Anti-ATG5/Apg5 Rabbit Monoclonal Antibody
Cat # M00240
Two the supplier anti-ATG5 antibodies, A00240 and M00240, have supplied Western blot image descriptions. A00240 is shown with rat spleen tissue lysate at 1 and 2 μg/mL. M00240 is shown with human HeLa, 293T, MCF-7, and K562 whole cell lysates; rat liver tissue and C6 whole cell lysates; and mouse liver tissue and NIH/3T3 whole cell lysates.
Which to pick: A00240 has a supplied rat spleen example. M00240 has supplied examples for the listed human cell lines, rat liver and C6 cells, and mouse liver and NIH/3T3 cells. Check the antibody cards for reactivity with your intended sample.
Source: BosterBio ATG5 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9H1Y0.
- Human Protein Atlas. ATG5 tissue expression.
- PMC6796855 — target-verified WB comparison
- PMC8679033 — target-verified WB comparison
- PMC8595469 — target-verified WB comparison