This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
ULK1 · Western blot design guide
Design a Western Blot for ULK1
Real validated ULK1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ULK1 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
ULK1 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 | ~112.6 kDa | |
| Observed band | ~120 kDa | |
| Gel | 8–10% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation-induced mobility shift | |
| Regulation | IFN-γ-induced | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated ULK1 Western Blot Protocols
Literature-validated Western blot parameters for ULK1 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human A549 , Lane 2: human THP-1 , Lane 3: rat brain , Lane 4: mouse brain . After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-ULK1 antigen affinity purified polyclonal antibody (Catalog # A00584-1) 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 1:10000 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 ULK1 at approximately 120KD. The expected band size for ULK1 is at 120KD |
| Gel % | 8–10% |
| Load | 50ug |
| 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, 1:10000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 120 kDa |
Section 2
What Is the Expected ULK1 Western Blot Band Size?
ULK1 has a 112.6 kDa predicted backbone but runs at ~120 kDa on Western blots, mainly due to extensive phosphorylation across its 18 annotated modification sites.
| single band around 120 kDa | reflects full-length ULK1's 112.6 kDa predicted core mass shifted upward by its extensive phosphorylation (18 annotated modification sites) |
| band running above the 112.6 kDa predicted mass | not a distinct proteoform but the expected mobility effect of heavy phosphorylation, including the AMPK-dependent Ser317 site |
| doublet or fuzzy band edge rather than one crisp line | different phosphorylation states of ULK1 (e.g. basal versus AMPK-activated) can resolve as slightly separated species |
| band present in standard whole-cell lysate | ULK1 is cytoplasmic/cytosolic, not secreted, so it is retained in the cell and detected directly in lysate without enrichment |
| no smaller cleaved fragment accompanying the main band | no signal peptide or propeptide is annotated, so full-length ULK1 is already the mature form with no expected maturation cleavage |
| only one band size, no isoform-driven ladder | only a single isoform is annotated for ULK1, so no additional isoform-specific bands are expected |
💡Expected ULK1 appearanceExpect a single band at approximately 120 kDa on reducing SDS-PAGE, running above the 112.6 kDa predicted mass for full-length ULK1 due to its extensive phosphorylation, with no cleavage or glycosylation-driven shifts.
| predicted mass (112.6 kDa, 1050 aa) | sets the baseline unmodified backbone size before any mobility shift is applied |
| extensive phosphorylation (18 annotated sites, including AMPK-mediated Ser317) | retards SDS-PAGE migration, pushing the apparent band up toward the observed ~120 kDa |
| lysine acetylation (Lys162) | adds a further, smaller charge-based contribution to apparent mobility shift |
| single annotated isoform | no isoform-driven band-size variation is expected; only one full-length species should be seen |
| absence of signal peptide or propeptide | rules out a smaller mature/cleaved product; the observed band represents the full-length precursor itself |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | extensive phosphorylation and acetylation across 18 annotated sites, including AMPK-driven Ser317, retard migration and push the band toward ~120 kDa versus the 112.6 kDa predicted mass | confirm the band matches the expected ~120 kDa empirical size, and run a phosphatase-treated lysate lane in parallel to confirm the shift collapses toward 112.6 kDa |
| Band lower than expected | loss of phosphorylation during sample handling, from insufficient phosphatase inhibitors, can collapse the band back toward the unmodified 112.6 kDa core | add phosphatase inhibitors to the lysis buffer and minimize time at room temperature before denaturing the sample |
| Broad smear instead of sharp band | heterogeneous phosphorylation states across the 18 modification sites produce a mixed population of slightly different masses | process lysate quickly and cold, and compare against a phosphatase-treated control lane to sharpen the band |
| Multiple bands | distinct phosphorylation states, such as basal versus AMPK-activated Ser317 phosphorylation, can resolve as closely spaced bands even with only one isoform annotated | standardize the metabolic or starvation state of cells before lysis and compare treated versus untreated samples to see if the extra band tracks with phosphorylation status |
| Weak or no signal | ULK1 phosphorylation and expression levels shift with autophagy-inducing conditions, and an antibody targeting a modified region may miss unmodified or differently phosphorylated pools | increase total protein loaded, induce autophagy (e.g. starvation) to boost the target signal, and confirm the antibody epitope relative to the annotated modification sites |
| Fragments below expected size | no propeptide or signal peptide is annotated for ULK1, so smaller fragments indicate proteolytic degradation during lysis rather than expected processing | include protease inhibitors in the lysis buffer, keep samples cold, and process lysates promptly to avoid degradation artifacts |
Experiment evidence
Western blot images showing ULK1 band
A00584-1 · ULK1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ULK1 Western blot
🧪For positive controls for ULK1 in Western blot, you can use whole-cell lysate from a common cell line such as HeLa or HEK293, since no tissue-specific HPA expression data is available and ULK1 is a widely expressed cytoplasmic kinase.
Positive control: HeLa or HEK293 cell lysate
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin alongside a total-protein stain (stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: No HPA tissue expression data exists for ULK1, so no clean negative tissue can be selected, and since it is a low-abundance cytoplasmic/preautophagosomal kinase, consider starvation or rapamycin treatment to boost signal and pair with siRNA/KO for a specificity control.
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 ULK1 Western Blot Tips
Deeper troubleshooting and optimisation questions for ULK1, answered from its protein features.
Why does ULK1 run near 120 kDa instead of 112.6 kDa?
ULK1's predicted mass is 112.6 kDa, but it carries 18 modified residues (mostly phosphorylation and acetylation), which retard mobility on SDS-PAGE. This is a common feature of heavily phosphorylated proteins and explains the observed ~120 kDa band. Treat this as a normal PTM-driven shift rather than degradation or an isoform difference.
Should multiple ULK1 isoforms be expected on blot?
UniProt lists only one ULK1 isoform, so a single band near 120 kDa is expected on a clean blot. Additional bands are more likely phospho-shifted species, proteolytic fragments, or antibody cross-reactivity than genuine isoforms. Validate with a knockdown or knockout control lane if extra bands appear consistently.
How does starvation affect ULK1 localization and phosphorylation?
Per UniProt, under starvation ULK1 relocalizes from cytosol to punctate preautophagosomal isolation-membrane structures as autophagy is induced. This shift is accompanied by regulatory phosphorylation changes rather than large changes in total protein amount, so induction is better tracked with phospho-specific ULK1 antibodies or fractionation than total-protein blot intensity alone.
What blocking buffer works best for ULK1?
ULK1 carries 18 modified residues and is commonly probed with phospho-specific antibodies; casein-based milk blockers contain phosphoproteins that can raise background with phospho-antibodies. Use BSA (3-5%) for blocking and antibody dilution when probing phosphorylated ULK1 sites. Milk is acceptable when using a total-ULK1 antibody that is not phospho-specific.
What transfer method to use for ULK1 Western blot?
ULK1 is a large protein (112.6 kDa predicted, ~120 kDa observed), so use wet/tank transfer with a low-methanol (10%) buffer, extended transfer time, and PVDF membrane. These conditions favor efficient transfer of this high-molecular-weight cytosolic kinase out of the gel compared to standard semi-dry protocols.
How to quantify ULK1 given its phosphorylation status?
ULK1 activity is governed largely by phosphorylation state across its 18 modified residues rather than large swings in total abundance. Quantify total ULK1 with a loading control to assess expression changes, and pair this with phospho-specific antibodies at known regulatory sites to assess activation state, rather than relying on total-protein intensity alone.
What explains unexpected extra bands on ULK1 blots?
ULK1 forms a complex with ATG13, RB1CC1/FIP200, and ATG101, and interacts with GABARAP/GABARAPL2; incomplete denaturation could leave co-migrating partners. Extra bands may also reflect phospho-shifted species arising from its 18 modified residues, or degradation of this large 112.6 kDa protein. Confirm specificity with a knockdown or knockout control lane.
Boster reagents
Best ULK1 Western Blot Antibodies
BosterBio's ULK1 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-ULK1 Antibody Picoband®
Cat # A00584-1
Our recommended anti-ULK1 antibodies represent top-performing, extensively cited reagents, rigorously validated by orthogonal methods including negative-tissue controls and complementary techniques, ensuring reliable, reproducible Western blot detection of ULK1.
Which to pick: Only one anti-ULK1 antibody is listed, A00584-1, which includes an authentic Western blot validation image showing specific ULK1 detection, making it the clear choice for your ULK1 Western blot experiments.
Source: BosterBio ULK1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O75385.
- Human Protein Atlas. ULK1 tissue expression.