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- Table of Contents
ATG12 / Ubiquitin-like protein ATG12 · Western blot design guide
Design a Western Blot for ATG12
Real validated ATG12 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ATG12 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
ATG12 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 | ~15.1 kDa | |
| Gel | 15% (standard starting point) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Oral mucosa (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Acetylated | |
| Caveat | ATG5 conjugate | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated ATG12 Western Blot Protocols
The A00820 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human brain tissue lysate (catalog A00820) |
| Gel % | 15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.2 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A00820 · (A) 0.5, and (B) 1 μg/mL (catalog A00820) |
| 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 ATG12 Western Blot Band Size?
Free ATG12 is predicted at 15.1 kDa; isoforms and ATG5 conjugation could affect the pattern, but their migration has not been demonstrated here.
| Band near 15.1 kDa | Consistent with free ATG12 at its predicted mass; confirm identity with controls |
| Band above 15.1 kDa | Could represent the ATG12–ATG5 conjugate; its apparent mass is not supplied |
| Several bands at different positions | Could include isoforms 1 and 2 or the ATG12–ATG5 conjugate; band identities need confirmation |
| Faint band near 15.1 kDa with a higher band | Free and ATG5-conjugated ATG12 could coexist |
💡Expected ATG12 appearanceFree ATG12 has a predicted mass of 15.1 kDa, but no empirical band size is supplied; confirm any band near that size or higher with band-identity controls.
| Predicted ATG12 mass | Places free ATG12 near 15.1 kDa, without establishing its observed migration |
| Isoform 1 | May differ in size from isoform 2; its mass and migration are not supplied |
| Isoform 2 | May differ in size from isoform 1; its mass and migration are not supplied |
| ATG12–ATG5 conjugation | Can produce a higher band than free ATG12; its apparent mass is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | ATG12 also associates with a peripheral membrane compartment | Check both soluble and membrane fractions and include a positive lysate control |
| Band higher than expected | ATG12 forms a conjugate with ATG5 | Probe for ATG5 and compare free and conjugated ATG12 signals |
| Band lower than expected | Isoform size differences are possible but unquantified | Compare an independent ATG12 antibody or use ATG12 depletion to verify identity |
| Multiple bands | ATG12 has isoforms 1 and 2 and forms an ATG5 conjugate | Use ATG12 depletion and ATG5 probing to assign the bands |
| Weak or no signal | Free ATG12 may be reduced when ATG12 is conjugated to ATG5 | Inspect higher bands and check antibody performance with a positive control |
Experiment evidence
Western blot images showing ATG12 band
A00820 · ATG12
A00820-2 · ATG12
M00820-2 · ATG12
M00820 · ATG12
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ATG12 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ATG12 in Western blot, you can use adrenal gland lysate, which HPA rates High.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Oral mucosa (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: Oral mucosa is HPA Not detected, but confirm the negative result in your lysate.
HPA tissue expression evidence for ATG12
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 → |
| Cerebral cortex | neuropil | High | Protein (IHC) | HPA → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular cells | High | Protein (IHC) | HPA → |
| Kidney | cells in tubules | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Oral mucosa | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | neuronal cells | Low | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Low | Protein (IHC) | HPA → |
| Hippocampus | neuronal cells | Low | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced ATG12 Western Blot Tips
Deeper troubleshooting and optimisation questions for ATG12, answered from its protein features.
How should ATG12 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 isoform 2 produce a smaller band?
Isoforms · Possibly. In isoform 2, UniProt residues 56–74 are replaced and residues 75–140 are missing. It is shorter than the 140-residue canonical sequence, but these features alone do not establish a visible band or its apparent mass.
Choose an epitope within UniProt residues 1–55, which precede the isoform 2 sequence change. An epitope within canonical residues 75–140 would be absent from isoform 2.
Choose an epitope within UniProt residues 1–55, which precede the isoform 2 sequence change. An epitope within canonical residues 75–140 would be absent from isoform 2.
Do annotated modifications prove a band shift?
PTM · The linked UniProt record describes protein features. A modification annotation alone does not demonstrate a visible shift; retain any condition or experimental qualifier attached to it.
Does this guide establish induction of ATG12?
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 ATG12?
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 A00820 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 ATG12 bands be quantified?
Quantitation · Quantify candidate free ATG12 and ATG12–ATG5 conjugate bands separately, stating which species each measurement represents. ATG12 conjugation means a single band may not represent all detectable ATG12.
How should a band above 15.1 kDa be interpreted?
Interpretation · 15.1 kDa is the predicted mass of canonical ATG12. ATG12 forms a conjugate with ATG5, so a higher band could represent that conjugate. The supplied features give no observed band position; mass alone cannot identify it.
Evaluate the 15.1 kDa canonical protein and any higher ATG12-reactive band separately. ATG12 forms an ATG5 conjugate, but the supplied features do not establish where either species runs on a blot.
Consider the shorter isoform 2 for a lower band and the ATG5 conjugate for a higher band. UniProt lists both isoforms and ATG5 conjugation, but neither feature alone identifies an observed band.
ATG12 is listed in the cytoplasm and at the preautophagosomal structure membrane as a peripheral membrane protein. TECPR1 recruits the ATG12–ATG5 conjugate to the autolysosomal membrane, so membrane-associated signal is consistent with these features.
Evaluate the 15.1 kDa canonical protein and any higher ATG12-reactive band separately. ATG12 forms an ATG5 conjugate, but the supplied features do not establish where either species runs on a blot.
Consider the shorter isoform 2 for a lower band and the ATG5 conjugate for a higher band. UniProt lists both isoforms and ATG5 conjugation, but neither feature alone identifies an observed band.
ATG12 is listed in the cytoplasm and at the preautophagosomal structure membrane as a peripheral membrane protein. TECPR1 recruits the ATG12–ATG5 conjugate to the autolysosomal membrane, so membrane-associated signal is consistent with these features.
Boster reagents
ATG12 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-Ubiquitin-like protein ATG12 ATG12 Antibody
Cat # A00820
Real WB data
Anti-ATG12 Antibody Picoband®
Cat # A00820-2
Real WB data
Anti-Apg12(Atg12) Rabbit Monoclonal Antibody
Cat # M00820-2
Real WB data
Anti-Apg12 (Atg12) Rabbit Monoclonal Antibody
Cat # M00820
All four listed anti-ATG12 antibodies have WB images from human samples. A00820-2 shows a band near 48 kDa, while its caption gives 15 kDa as the expected size. The supplied evidence does not resolve this discrepancy.
Which to pick: A00820 lists human, mouse, and rat reactivity, but its WB image uses human brain lysate. For human cell lysates, A00820-2 shows PC-3, COLO 320, HeLa, and HepG2; M00820-2 shows 293; M00820 shows HepG2. Consider A00820-2’s band-size discrepancy.
Source: BosterBio ATG12 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O94817.
- Human Protein Atlas. ATG12 tissue expression.
- PMC6985262 — target-verified WB comparison
- PMC5386389 — target-verified WB comparison
- PMC10531886 — target-verified WB comparison