ULK1 Antibody

What is ULK1 and why is it significant in autophagy research?

ULK1 (Unc-51 Like Autophagy Activating Kinase 1) is a serine/threonine protein kinase that plays a crucial role in the initiation of autophagy. It functions as an essential component in autophagosome formation by forming a stable complex with ATG13 and focal adhesion kinase (FAK) family interacting protein of 200 kDa (FIP 200) . ULK1 acts upstream of phosphatidylinositol 3-kinase PIK3C3 to regulate the formation of autophagophores, the precursors of autophagosomes .

The significance of ULK1 in research stems from its position as a convergence point for multiple regulatory signals controlling autophagy. ULK1 mediates crosstalk between two major cellular nutrient sensors: it is negatively regulated by mTORC1 and positively regulated by AMPK during nutrient starvation . Furthermore, ULK1 has been implicated in various diseases including cancer, neurodegenerative disorders, and metabolic conditions, making it an important target for therapeutic intervention strategies .

What is the molecular weight range for ULK1 detection in Western blot applications?

When detecting ULK1 using Western blot, researchers should expect to observe bands in the following ranges:

The calculated molecular weight of ULK1 is approximately 112-113 kDa , but post-translational modifications often result in the protein migrating at a higher apparent molecular weight. This variation in observed molecular weight may reflect different phosphorylation states or other modifications of ULK1, which are particularly relevant during autophagy induction .

What application methods are validated for ULK1 antibodies?

ULK1 antibodies have been validated for multiple experimental applications, with varying recommended dilutions:

It's important to note that each antibody should be titrated in your specific experimental system to achieve optimal results . Published literature can provide precedent for successful applications; for example, over 65 publications have validated the use of antibody 20986-1-AP for Western blot applications .

How should I optimize sample preparation for ULK1 Western blot analysis?

For optimal ULK1 detection by Western blot, consider the following methodological approach:

• Lysis buffer selection: Use a buffer containing phosphatase inhibitors to preserve phosphorylation states, especially when studying ULK1 activation. RIPA buffer supplemented with protease and phosphatase inhibitor cocktails is commonly used .

• Protein concentration measurement: Ensure equal loading (typically 20-40 μg total protein per lane) for consistent results.

• Denaturation conditions: Heat samples at 95°C for 5 minutes in Laemmli buffer containing SDS and β-mercaptoethanol to fully denature ULK1.

• Gel selection: Use 8% or 10% SDS-PAGE gels to provide good resolution in the 100-150 kDa range where ULK1 migrates .

• Transfer conditions: Opt for wet transfer methods with 10% methanol for proteins of this size (>100 kDa). Transfer at low voltage (30V) overnight at 4°C for improved transfer of large proteins.

• Blocking conditions: Block membranes with 5% non-fat dry milk or BSA in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature.

• Antibody incubation: Incubate with primary ULK1 antibody (at recommended dilution) overnight at 4°C, followed by appropriate HRP-conjugated secondary antibody .

Multiple positive control samples have been validated for ULK1 detection, including HEK-293T cells, HepG2 cells, HeLa cells, and mouse skeletal muscle tissue .

What strategies can be employed to detect phosphorylated forms of ULK1?

Detecting phosphorylated ULK1 requires specific methodological considerations:

• Phospho-specific antibodies: Use antibodies specifically targeting phosphorylated residues of interest. For example, antibodies targeting phospho-S556 (ab203207) have been validated for detecting ULK1 phosphorylated by AMPK .

• Sample preparation: Immediately lyse cells in buffer containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate, and β-glycerophosphate) to preserve phosphorylation states.

• Positive controls: Include samples from cells treated with rapamycin (which inhibits mTORC1, leading to reduced phosphorylation at S757) or AICAR/glucose starvation (which activates AMPK, leading to increased phosphorylation at S317, S555, and S777) .

• Validation methods:

  • Use λ-phosphatase treatment of duplicate samples as a negative control

  • Compare wild-type cells with those expressing ULK1 phosphorylation-site mutants

  • Pair phospho-specific antibody detection with total ULK1 detection for normalization

• Phosphorylation dynamics: Monitor time-dependent changes in ULK1 phosphorylation status. For example, upon amino acid starvation, phosphorylation of ULK1 at S757 (the mTORC1 target site) decreases, although more slowly than the dephosphorylation of S6K (another mTORC1 substrate) .

How can I effectively design co-immunoprecipitation experiments to study ULK1 protein complexes?

To study ULK1 protein complexes through co-immunoprecipitation:

• Antibody selection: Choose ULK1 antibodies validated for immunoprecipitation. The rabbit monoclonal antibody #8054 from Cell Signaling Technology has been validated for this application .

• Lysis conditions: Use mild lysis buffers (containing 0.5% NP-40 or 1% Triton X-100) to preserve protein-protein interactions. Avoid harsh detergents like SDS that may disrupt complexes.

• Preclearing: Preclear lysates with protein A/G beads to reduce non-specific binding.

• Controls: Include:

  • IgG control (same species as ULK1 antibody)

  • Input samples (5-10% of the lysate used for IP)

  • When possible, ULK1 knockout/knockdown samples as negative controls

• Detection strategy: For detecting interaction partners, consider:

  • Probing for known ULK1 interactors (ATG13, FIP200, ATG101)

  • Examining specific components like Beclin-1 and ATG14L, which form complexes with ULK1 during autophagy induction

• Experimental validation: Research has shown that ATG14L facilitates ULK1-Beclin-1 interaction. Co-immunoprecipitation experiments revealed that Beclin-1 pulled down ULK1 only when co-transfected with ATG14L, while ATG14L could pull down ULK1 in the absence of Beclin-1, suggesting ATG14L may recruit Beclin-1 to ULK1 for phosphorylation .

Why might I observe multiple bands or varied molecular weights when detecting ULK1?

Multiple bands or varied molecular weights in ULK1 detection can result from several factors:

• Post-translational modifications: ULK1 undergoes extensive phosphorylation at multiple sites by AMPK and mTORC1, which can alter its migration pattern. The calculated molecular weight is approximately 112-113 kDa, but the observed molecular weight ranges from 113-150 kDa across different studies .

• Isoforms and splice variants: While less common for ULK1, potential alternative splicing could generate different protein products.

• Proteolytic degradation: Insufficient protease inhibition during sample preparation may result in degradation products appearing as lower molecular weight bands.

• Cross-reactivity: Some antibodies may cross-react with the paralog ULK2, which shares significant sequence homology with ULK1 .

• Technical factors: Inconsistent SDS-PAGE conditions, transfer efficiency variations, or overexposure during imaging can affect band appearance.

To address these issues:

• Use fresh lysates with complete protease inhibitor cocktails

• Compare results with published literature for your specific antibody

• Validate bands using ULK1 knockout/knockdown controls

• Consider using both monoclonal and polyclonal antibodies to confirm specificity

What are effective strategies for optimizing ULK1 immunofluorescence staining?

For optimal ULK1 immunofluorescence staining, consider the following strategies:

• Fixation method optimization:

  • 4% paraformaldehyde (10-15 minutes) preserves cellular architecture

  • Methanol fixation (100% methanol at -20°C for 10 minutes) may better expose some epitopes

  • Test both methods to determine which works best with your specific antibody

• Permeabilization conditions:

  • 0.1-0.3% Triton X-100 in PBS for 10 minutes

  • 0.1% saponin may be gentler for preserving membrane structures

• Blocking optimization:

  • 5-10% normal serum (from the same species as the secondary antibody)

  • 1-3% BSA in PBS

  • Include 0.1% Tween-20 to reduce background

• Antibody dilution optimization:

  • Start with manufacturer's recommended range (typically 1:50-1:500)

  • Prepare a dilution series to determine optimal signal-to-noise ratio

• Signal amplification:

  • Consider tyramide signal amplification for low-abundance targets

  • Use high-sensitivity detection systems for challenging applications

• Validated positive controls:

  • HeLa cells have been consistently validated for ULK1 immunofluorescence

  • Include starvation conditions (EBSS or serum-free media) to induce autophagy and observe ULK1 puncta formation

• Co-staining strategies:

  • Co-stain with autophagosome markers (LC3B) to validate ULK1 localization

  • Use markers for early autophagosomal structures to confirm physiological relevance

How can I validate the specificity of my ULK1 antibody?

Validating ULK1 antibody specificity requires a multi-faceted approach:

• Genetic validation:

  • Use ULK1 knockout (KO) or knockdown (KD) samples as negative controls

  • The literature documents several publications using ULK1 KO/KD validation approaches

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide prior to application

  • Specific signals should be blocked by the competing peptide

• Recombinant protein expression:

  • Overexpress tagged ULK1 (GFP-ULK1, Flag-ULK1) and confirm detection at the expected size

  • Verify detection using antibodies against both ULK1 and the tag

• Phosphatase treatment:

  • When using phospho-specific antibodies, treat samples with λ-phosphatase

  • Phospho-specific signals should disappear after phosphatase treatment

• Comparison across multiple antibodies:

  • Use antibodies targeting different epitopes of ULK1

  • Consistent detection patterns increase confidence in specificity

• Correlation with functional readouts:

  • Verify that ULK1 activation (detected by antibodies) correlates with downstream autophagy markers

  • For example, ULK1 activation should lead to increased Beclin-1 phosphorylation at Serine 14

• Mass spectrometry validation:

  • For advanced validation, immunoprecipitate ULK1 and confirm identity by mass spectrometry

How can I study the ULK1-mediated phosphorylation of Beclin-1 and its impact on autophagy?

Studying ULK1-mediated phosphorylation of Beclin-1 requires specific methodological approaches:

• Detection of phosphorylated Beclin-1:

  • Use phospho-specific antibodies targeting Beclin-1 Serine 14 (S15 in humans), which is the primary ULK1 phosphorylation site

  • Confirm specificity using Beclin-1 S14A mutant, which abolishes ULK1-mediated phosphorylation

• In vitro kinase assays:

  • Use purified ULK1 (from insect cells) and recombinant Beclin-1 (from E. coli)

  • Include ATG14L, as research shows ATG14L-containing Beclin-1 is more efficiently phosphorylated by ULK1 than Beclin-1 alone

  • Analyze phosphorylation using radiolabeled ATP (γ-32P-ATP) or phospho-specific antibodies

• Cellular studies:

  • Compare Beclin-1 phosphorylation in wild-type versus ULK1 knockout/knockdown cells

  • Induce ULK1 activation through amino acid starvation and monitor the time course of Beclin-1 phosphorylation

  • As shown in the literature, endogenous Beclin-1 phosphorylation increases upon amino acid starvation, while phosphatase treatment abolishes the phospho-S14 signal

• Structure-function analysis:

  • Generate Beclin-1 phosphomimetic (S14D/E) and phospho-deficient (S14A) mutants

  • Assess their impact on VPS34 complex formation and autophagy induction

• Functional consequences:

  • Measure VPS34 kinase activity in immunoprecipitated ATG14L-containing complexes

  • Research has demonstrated that ectopic expression of ULK1 dramatically increases the activity of ATG14L-containing VPS34 complexes

What methods can be used to study the regulation of ULK1 by upstream kinases mTORC1 and AMPK?

To study the regulation of ULK1 by mTORC1 and AMPK:

• Phosphorylation-site analysis:

  • mTORC1 phosphorylates ULK1 at S757 (inhibitory)

  • AMPK phosphorylates ULK1 at multiple sites including S317, S555, and S777 (activating)

  • Use phospho-specific antibodies targeting these sites to monitor the phosphorylation status under different conditions

• Pharmacological approaches:

  • Inhibit mTORC1 using rapamycin, Torin1, or amino acid starvation

  • Activate AMPK using AICAR, metformin, or glucose deprivation

  • Monitor changes in ULK1 phosphorylation status at specific residues

• Genetic manipulation:

  • Use cells expressing phospho-mutant versions of ULK1 (S757A, S317A/S555A/S777A)

  • Employ mTORC1 component knockouts (RAPTOR) or AMPK knockouts

  • Assess impacts on ULK1 activation and downstream autophagy

• Time-course experiments:

  • Research shows that upon amino acid withdrawal, phosphorylation of ULK1 at S757 (the mTORC1 target site) decreases, although more slowly than other mTORC1 substrates like S6K

  • Monitor the kinetics of these changes to understand the temporal regulation

• Co-immunoprecipitation studies:

  • Examine physical interactions between ULK1 and components of mTORC1 or AMPK complexes

  • Determine how these interactions change under different nutritional conditions

• Functional readouts:

  • Measure autophagy markers (LC3-II, p62 degradation)

  • Assess ULK1 kinase activity toward substrates like Beclin-1

  • Quantify autophagosome formation using fluorescence microscopy

How can I investigate ULK1's role in diseases such as neurodegeneration and cancer?

Investigating ULK1's role in disease contexts requires specialized approaches:

• Expression analysis in disease tissues:

  • Use ULK1 antibodies for immunohistochemistry on disease versus normal tissues

  • Multiple ULK1 antibodies have been validated for human tissues, including skeletal muscle, liver, and liver cancer tissue

  • Research suggests ULK1 represents a potential novel prognostic biomarker for hepatocellular carcinoma (HCC) patients

• Genetic association studies:

  • Analyze ULK1 mutations or polymorphisms in patient cohorts

  • Consider ULK1's association with neurodegeneration with brain iron accumulation

• Disease models:

  • Generate ULK1 knockout or transgenic animal models

  • Examine tissue-specific ULK1 deletion (e.g., neuron-specific or hepatocyte-specific)

  • Assess disease progression and autophagy status in these models

• Cellular disease models:

  • Use patient-derived cells or genetically modified cells expressing disease-associated mutations

  • Examine ULK1 activation and autophagy in response to disease-relevant stressors

• Therapeutic targeting approaches:

  • Test ULK1 activators or inhibitors in disease models

  • Assess impact on disease progression, autophagy flux, and cellular homeostasis

• Interaction with disease-associated proteins:

  • In neurodegenerative diseases, examine ULK1's relationship with proteins like tau, α-synuclein, or huntingtin

  • In cancer, investigate ULK1's interactions with tumor suppressors or oncogenes

• Stress response studies:

  • Evaluate how ULK1-mediated autophagy responds to disease-relevant stresses

  • For example, in cancer, examine nutrient limitation, hypoxia, or chemotherapy treatment

How should I interpret changes in ULK1 localization and activity during autophagy induction?

Interpreting ULK1 changes during autophagy requires understanding several key aspects:

What are the key differences between ULK1 and its paralog ULK2 that researchers should consider?

When investigating ULK proteins, researchers should consider these key differences between ULK1 and ULK2:

• Structural similarities and differences:

  • Both belong to the protein kinase superfamily, Ser/Thr protein kinase family, and APG1/unc-51/ULK subfamily

  • Both contain an N-terminal kinase domain, central proline/serine-rich domain, and conserved C-terminal domain

  • Sequence variations may affect antibody cross-reactivity and substrate specificity

• Expression patterns:

  • Both are widely expressed, but may show tissue-specific expression differences

  • Consider potential compensation mechanisms in knockout models

• Functional redundancy and specificity:

  • ULK1 and ULK2 show partial functional redundancy in autophagy

  • Single knockouts of either gene often show mild phenotypes compared to double knockouts

  • ULK1 appears to have a more prominent role in starvation-induced autophagy in most cell types

• Antibody considerations:

  • Check for potential cross-reactivity of ULK1 antibodies with ULK2

  • When possible, use antibodies targeting unique epitopes to distinguish between paralogs

  • Validate specificity using individual knockout controls

• Experimental approaches:

  • Consider using siRNA targeting both ULK1 and ULK2 to fully inhibit the ULK pathway

  • In knockdown/knockout experiments, assess compensation by the remaining paralog

  • When studying ULK1-specific functions, validate with rescue experiments using ULK1 but not ULK2

• Evolutionary conservation:

  • Both proteins evolved from the C. elegans gene unc-51, where mutants exhibited abnormal axonal extension and growth

  • Consider evolutionary aspects when using different model organisms

What emerging technologies and approaches show promise for advancing ULK1 research?

Several cutting-edge approaches are advancing ULK1 research:

• CRISPR/Cas9 genome editing:

  • Generation of endogenously tagged ULK1 (e.g., GFP-ULK1 knock-in)

  • Creation of phospho-mutant knock-in models

  • Precise editing of ULK1 regulatory regions to study transcriptional control

• Proximity labeling proteomics:

  • BioID or APEX2 fusion with ULK1 to identify proximal interacting proteins

  • TurboID for rapid labeling of transient interactions during autophagy initiation

  • Identification of context-specific ULK1 interaction networks

• Advanced imaging techniques:

  • Super-resolution microscopy (STORM, PALM) for detailed visualization of ULK1 complexes

  • Live-cell imaging with fluorescent ULK1 to track dynamics in real-time

  • Correlative light and electron microscopy (CLEM) to connect ULK1 localization with ultrastructural changes

• Structural biology approaches:

  • Cryo-EM structures of ULK1 complexes

  • Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

  • Structure-guided development of specific inhibitors or activators

• Single-cell analysis:

  • Single-cell transcriptomics to evaluate ULK1 expression heterogeneity

  • Single-cell proteomics to measure ULK1 protein levels and modifications

  • Correlation of ULK1 status with autophagy markers at the single-cell level

• In vivo tools:

  • Development of transgenic mouse models with fluorescent reporters for ULK1 activity

  • Tissue-specific and inducible ULK1 manipulation systems

  • Optogenetic control of ULK1 activity for spatial and temporal precision

• Chemical biology approaches:

  • Development of highly specific ULK1 inhibitors for therapeutic applications

  • Activity-based probes to measure ULK1 kinase activity in situ

  • Degradation-targeting chimeras (PROTACs) for selective ULK1 degradation