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ATG13 · Western blot design guide
Design a Western Blot for ATG13
Real validated ATG13 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ATG13 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
ATG13 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 | ~56.6 kDa | |
| Observed band | ~56 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Alternative splicing isoforms | |
| Regulation | LPS-induced | |
| Isoform | 5 isoform(s) |
Section 1
Real Curated ATG13 Western Blot Protocols
Literature-validated Western blot parameters for ATG13 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | Human Placenta at 50ug. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-KIAA0652 antigen affinity purified polyclonal antibody (Catalog # PB9480) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for KIAA0652 at approximately 56 kDa. The expected band size for KIAA0652 is at 56 kDa |
| Gel % | 10–12% |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 56 kDa |
Section 2
What Is the Expected ATG13 Western Blot Band Size?
ATG13 has a 56.6 kDa predicted mass matching its observed ~56 kDa band, with phosphorylation and its five splice isoforms as the main sources of minor band variation.
| single band at ~56 kDa | matches the predicted 56.6 kDa mass of unmodified ATG13, consistent with no signal peptide, propeptide, glycosylation, or disulfide cross-linking |
| slight upward shift or faint doublet near 56 kDa | reflects phosphorylation at Ser355 (by ULK1), Ser356 and Ser361, which can alter SDS-PAGE mobility |
| multiple bands across a range of molecular weights | ATG13 has five annotated splice isoforms, each capable of producing a distinct band |
| no additional band at roughly double the monomer mass | no inter-chain disulfide bonds are annotated, so ATG13 does not form a covalent homodimer; its ULK1-RB1CC1-ATG101 complex is non-covalent and dissociates under SDS-PAGE |
| band present in standard whole-cell or cytosolic lysate | ATG13 is a cytoplasmic/cytosolic protein rather than secreted, so it is expected in routine lysate preparations |
💡Expected ATG13 appearanceATG13 runs as a single band at approximately 56 kDa, matching both its 56.6 kDa predicted mass and the empirically observed ~56 kDa band, since it lacks glycosylation, disulfide bonds, and cleaved signal or propeptide sequences.
| predicted mass from UniProt | sets the baseline expectation of ~56.6 kDa, closely matching the empirically observed ~56 kDa band |
| absence of signal peptide or propeptide | no precursor-to-mature cleavage occurs, so no smaller processed fragment is expected below the full-length band |
| phosphorylation at Ser355 (by ULK1), Ser356 and Ser361 | can cause a modest upward mobility shift or a doublet near the main band on SDS-PAGE |
| five annotated splice isoforms | different isoforms may appear as additional bands running qualitatively higher or lower than the main band |
| absence of inter-chain disulfide bonds | no covalent homodimer band forms, despite ATG13 participating in the non-covalent ULK1-RB1CC1-ATG101 complex |
| absence of N-/O-glycosylation | no smearing or upward mass shift from glycan addition is expected |
| Situation | Likely cause | Next action |
|---|---|---|
| Multiple bands | presence of five annotated ATG13 splice isoforms combined with phosphorylation-shifted forms of the main band | compare band pattern against known isoform masses and use phosphatase treatment to collapse phospho-shifted bands |
| Band higher than expected | phosphorylation at Ser355 (ULK1 site), Ser356 and Ser361 slows migration on SDS-PAGE | treat lysate with lambda phosphatase before loading to confirm the shift is phosphorylation-dependent |
| Weak or no signal | ATG13 abundance and puncta localization change with autophagy/starvation status, and its tight complex with ULK1 and RB1CC1 can mask the epitope | induce autophagy by starvation before lysis and use a lysis buffer that fully disrupts the ULK1-RB1CC1-ATG101 complex |
| No band in lysate | insufficient enrichment of a low-abundance cytosolic regulatory protein in the chosen cell line or tissue | load more total protein and confirm baseline ATG13 expression in the model system before further troubleshooting |
| Fragments below expected size | proteolytic degradation during lysis, since ATG13 has no cleaved signal peptide or propeptide to account for smaller native fragments | add protease inhibitors and keep samples cold throughout lysis and sample preparation |
Experiment evidence
Western blot images showing ATG13 band
PB9480 · ATG13
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ATG13 Western blot
🧪For positive controls for ATG13 in Western blot, you can use whole-cell lysate from a standard human cell line such as HeLa or HEK293, since ATG13 is a core, broadly-expressed cytosolic autophagy component rather than a tissue-restricted protein.
Positive control: HeLa or HEK293 whole-cell lysate
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin as loading controls alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) to confirm equal loading.
⚠️Feasibility: No HPA tissue expression data are available for ATG13, and as a cytosolic/preautophagosomal core autophagy protein it is expected to be broadly expressed, so a clean tissue-based negative control is unlikely to exist—confirm antibody specificity with siRNA knockdown or a CRISPR KO line instead.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced ATG13 Western Blot Tips
Deeper troubleshooting and optimisation questions for ATG13, answered from its protein features.
Does the observed band match predicted ATG13 mass?
Yes. Predicted mass is 56.6 kDa and the observed band runs at approximately 56 kDa, showing minimal shift. This close agreement makes ATG13 a reliable single-band marker, though phosphorylation upon autophagy induction can cause slight upward mobility shifts.
Why does ATG13 show multiple bands on blots?
UniProt lists 5 annotated isoforms generated by alternative splicing. Depending on cell type and antibody epitope, one or more isoform bands may be detected near 56 kDa, so a secondary band does not necessarily indicate nonspecific binding.
How does starvation affect the ATG13 band pattern?
ATG13 carries 4 modified residues including phosphorylation and acetylation sites. Upon nutrient starvation, ULK1-complex-driven phosphorylation of ATG13 can produce a slower-migrating, slightly diffuse band compared to fed-state lysates.
What blocking buffer avoids phospho-epitope loss?
Because ATG13 is a phosphoprotein, use BSA-based blocking rather than milk if probing phospho-specific epitopes, since casein in milk can mask or interfere with phospho-antibody binding. Standard non-phospho detection tolerates either blocking agent.
What transfer method to use for ATG13 Western blot?
Standard wet or semi-dry transfer at 56 kDa works well; no signal peptide, propeptide, or heavy glycosylation is present to complicate transfer. Use PVDF membrane for optimal retention of this moderately sized cytosolic phosphoprotein.
Is ATG13 suitable for reliable quantitation?
Yes, since predicted and observed masses align closely (56.6 vs ~56 kDa) with no glycosylation or disulfide bonding to introduce variability, ATG13 typically resolves as a clean, quantifiable single band suitable for densitometric comparison across samples.
What causes unexpected higher molecular weight ATG13 bands?
ATG13 forms a stable complex with ULK1, RB1CC1, and ATG101. Incomplete denaturation or insufficient reducing conditions may leave residual complex association, producing higher apparent weight bands; ensure thorough boiling in SDS sample buffer to dissociate interactions.
Boster reagents
Best ATG13 Western Blot Antibodies
BosterBio's ATG13 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-KIAA0652/ATG13 Antibody Picoband®
Cat # PB9480
For ATG13 Western blotting, Boster's PB9480 antibody is supported by a documented WB image demonstrating specific detection, making it a practical, image-verified starting point for your experiment.
Which to pick: Only one ATG13 antibody is catalogued here: PB9480. It has an associated Western blot validation image, so it's the recommended choice by default since no alternative SKUs are available for comparison.
Source: BosterBio ATG13 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O75143.
- Human Protein Atlas. ATG13 tissue expression.