ULK1 Antibody, HRP conjugated

What is the target specificity of ULK1 Antibody, HRP conjugated?

ULK1 Antibody, HRP conjugated specifically targets the amino acid region 602-715 of human Serine/threonine-protein kinase ULK1. This polyclonal antibody is developed in rabbits using a recombinant human ULK1 protein fragment (602-715AA) as the immunogen . The antibody demonstrates high specificity for human ULK1, with >95% purity achieved through Protein G purification methods .

When selecting between different ULK1 antibodies, researchers should consider that several variants target different regions of the protein. For instance, some antibodies target amino acids 951-1050, 400-499, or 567-577, and these different epitope specificities may be crucial depending on your experimental goals . The 602-715 region represents a segment outside the kinase domain that may be important for protein-protein interactions in the autophagy pathway.

What applications are validated for ULK1 Antibody, HRP conjugated?

The primary validated application for ULK1 Antibody, HRP conjugated is ELISA (Enzyme-Linked Immunosorbent Assay) . The HRP conjugation provides direct enzymatic detection capability, eliminating the need for secondary antibody incubation steps in experimental protocols.

While ELISA is the manufacturer-validated application, researchers should note that similar antibodies targeting the same or nearby epitopes but with different conjugates or unconjugated formats have been successfully used in additional applications including:

• Western Blotting (WB)

• Immunofluorescence (IF)

• Immunocytochemistry (ICC)

• Flow Cytometry (FACS)

• Immunohistochemistry (IHC)

When adapting this HRP-conjugated antibody for applications beyond ELISA, empirical determination of optimal working dilutions and thorough validation is essential for reliable results.

What is the significance of ULK1 in autophagy research?

ULK1 (Unc-51 Like Kinase 1) plays a critical role as a serine/threonine kinase that functions as an upstream regulator in the autophagy pathway. It acts as both a downstream effector and negative regulator of mammalian target of rapamycin complex 1 (mTORC1) via interaction with RPTOR, establishing important regulatory feedback loops in autophagy .

The significance of ULK1 in research extends to multiple areas:

• ULK1 operates upstream of phosphatidylinositol 3-kinase PIK3C3 to regulate autophagosome formation

• It is activated via phosphorylation by AMPK and also regulates AMPK activity through mediating phosphorylation of AMPK subunits PRKAA1, PRKAB2, and PRKAG1

• ULK1 may phosphorylate multiple substrates including ATG13/KIAA0652, RPTOR, SESN2, and SQSTM1 to regulate autophagy

• Recent research has developed ULK1-Recruiting Chimeras (ULKRECs) that can direct ULK1 to specific cellular targets, demonstrating that localized ULK1 activity is sufficient to induce autophagosome formation

• ULK1 inhibition has shown promise in cancer immunotherapy research, particularly in LKB1 mutant lung cancer, where it can overcome compromised antigen presentation and restore antitumor immunity

This multifaceted role makes ULK1 a critical target for antibody-based detection in numerous autophagy-related research contexts.

What are the optimal experimental conditions for using ULK1 Antibody, HRP conjugated in ELISA?

When using ULK1 Antibody, HRP conjugated for ELISA applications, researchers should consider the following methodological guidelines:

• Dilution Optimization: The manufacturer advises that optimal working dilutions should be determined empirically for each specific experimental system . Generally, starting with a 1:1000 dilution and performing a dilution series (1:500, 1:1000, 1:2000, 1:5000) will help identify the optimal signal-to-noise ratio.

• Buffer Composition: The antibody is typically provided in a buffer containing preservatives (0.03% Proclin™300) that should be considered when designing experiments sensitive to preservatives .

• Blocking Protocol: For optimal results, use 5% non-fat dry milk or BSA in PBST (PBS + 0.1% Tween-20) for blocking non-specific binding sites. Block for 1-2 hours at room temperature.

• Control Inclusions: Always include both positive and negative controls:

  • Positive control: Recombinant human ULK1 protein or lysates from cells known to express high levels of ULK1

  • Negative control: Samples from ULK1 knockout cells or isotype control antibody at the same concentration

• Detection Parameters: Due to the HRP conjugation, direct detection using TMB or other appropriate HRP substrates is possible. The chromogenic reaction should be stopped with 2N H₂SO₄ after appropriate development time (typically 5-15 minutes), and absorbance measured at 450nm with reference wavelength at 620nm.

How can researchers validate the specificity of ULK1 Antibody, HRP conjugated?

Validating antibody specificity is crucial for ensuring reliable experimental results. For ULK1 Antibody, HRP conjugated, consider the following validation approaches:

• Genetic Validation:

  • Compare signals between wild-type cells and ULK1 knockout or knockdown cells

  • Perform overexpression experiments with tagged ULK1 constructs as positive controls

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess immunizing peptide (ULK1 amino acids 602-715)

  • A specific antibody will show reduced or eliminated signal when blocked with its cognate peptide

• Cross-Reactivity Assessment:

  • Test the antibody against closely related proteins like ULK2

  • Compare reactivity across species (although this antibody is specifically designed for human ULK1)

• Molecular Weight Verification:

  • In Western blot applications, verify that detected bands correspond to the expected molecular weight of ULK1 (approximately 112 kDa)

  • Look for phosphorylated forms that may show slight MW shifts

• Subcellular Localization:

  • In IF/ICC applications, confirm that staining patterns match the expected subcellular localization of ULK1, which typically shows cytoplasmic distribution with punctate structures during autophagy induction

A comprehensive validation approach utilizing multiple methods provides the strongest evidence for antibody specificity.

What controls should be included when studying ULK1-mediated autophagy?

When investigating ULK1-mediated autophagy using ULK1 Antibody, HRP conjugated, the following controls should be included:

• Autophagy Induction and Inhibition Controls:

  • Positive control: Treat cells with known autophagy inducers (e.g., rapamycin, starvation conditions)

  • Negative control: Treat cells with autophagy inhibitors (e.g., 3-methyladenine, bafilomycin A1)

  • These controls help establish the dynamic range of ULK1 activity in your system

• ULK1 Phosphorylation Status Controls:

  • AMPK activation control: AICAR or glucose starvation to promote activating ULK1 phosphorylation

  • mTOR activation control: Insulin or amino acid stimulation to promote inhibitory ULK1 phosphorylation

  • Parallel detection of phosphorylated ULK1 (particularly at Ser556) provides context for total ULK1 levels

• Pathway Controls:

  • Monitor downstream autophagy markers (LC3-I to LC3-II conversion, p62/SQSTM1 degradation)

  • Include ATG13 detection, a direct ULK1 substrate

  • Detection of AMPK and mTOR activity markers to correlate with ULK1 status

• Technical Controls for HRP-Conjugated Antibodies:

  • Enzyme activity control: Verify HRP activity using a direct substrate test

  • Background control: Include secondary-only controls when optimizing protocols

  • Quenching control: Test for potential peroxidase activity in your biological samples

This multi-level control strategy ensures reliable interpretation of ULK1-related data in autophagy research contexts.

How can ULK1 Antibody, HRP conjugated be used in cancer immunotherapy research?

Recent research has uncovered a significant role for ULK1 in cancer immunotherapy, particularly in the context of LKB1 mutant lung cancer. ULK1 Antibody, HRP conjugated can be utilized in several sophisticated experimental approaches in this field:

• Monitoring ULK1 Activity in Immune Checkpoint Inhibition Studies:
  Recent findings demonstrate that ULK1 inhibition can overcome compromised antigen presentation and restore antitumor immunity in LKB1 mutant lung cancer . Researchers can use ULK1 Antibody, HRP conjugated to:

  • Monitor ULK1 expression levels in tumor samples before and after treatment with ULK1 inhibitors like MRT68921

  • Correlate ULK1 levels with response to immunotherapy (e.g., anti-PD1 treatment)

  • Develop ELISA-based screening assays for patient stratification

• Investigating ULK1-Mediated Autophagy in Antigen Processing:
  ULK1 inhibition enhances immunoproteasome activity, which is crucial for antigen processing . Experimental approaches include:

  • Quantifying ULK1 expression in antigen-presenting cells under various conditions

  • Correlating ULK1 levels with immunoproteasome activity markers

  • Measuring changes in antigen presentation efficiency relative to ULK1 activity

• ULK1 Status as a Biomarker in Combination Therapies:
  The combination of ULK1 inhibition (using MRT68921) with anti-PD1 therapy has shown enhanced efficacy in tumor regression . ULK1 Antibody, HRP conjugated can be used to:

  • Develop ELISA-based prognostic or predictive assays for patient selection

  • Monitor treatment response through serial sampling

  • Establish thresholds of ULK1 activity that correlate with therapeutic outcomes

This advanced application highlights the translational potential of ULK1 research in precision oncology.

What approaches can be used to study ULK1 activation mechanisms using this antibody?

Understanding the complex activation mechanisms of ULK1 requires sophisticated experimental approaches. ULK1 Antibody, HRP conjugated can be incorporated into the following methodological strategies:

• Phosphorylation-State Analysis:
  ULK1 activity is regulated by various phosphorylation events, including activating phosphorylation by AMPK and inhibitory phosphorylation by mTORC1 . Researchers can:

  • Combine ULK1 Antibody, HRP conjugated with phospho-specific antibodies in multiplexed ELISA

  • Correlate total ULK1 levels with phosphorylation status under different conditions

  • Establish activation/inhibition ratios by normalizing phospho-ULK1 to total ULK1

• ULK1 Complex Formation Analysis:
  ULK1 functions in a complex with ATG13, FIP200, and ATG101. Researchers can:

  • Use ULK1 Antibody, HRP conjugated in sandwich ELISA formats to detect complex formation

  • Perform pull-down assays followed by detection of complex components

  • Study the dynamics of complex assembly/disassembly under autophagy-inducing conditions

• ULK1 Substrate Phosphorylation Profiling:
  ULK1 phosphorylates multiple substrates including ATG13, RPTOR, AMPK subunits, SESN2, and SQSTM1 . Approaches include:

  • Developing in vitro kinase assays using purified ULK1 and candidate substrates

  • Correlating ULK1 levels with substrate phosphorylation status

  • Comparing substrate phosphorylation patterns in the presence of ULK1 inhibitors

• Spatial Organization of ULK1 Activity:
  The localization of ULK1 is critical for autophagosome formation. Advanced approaches include:

  • Studying ULK1 recruitment to specific subcellular locations

  • Analyzing the effects of ULKRECs (ULK1-Recruiting Chimeras) on targeted autophagy

  • Developing ELISA-based assays for compartment-specific ULK1 activity

These methodological approaches enable detailed investigation of ULK1 activation mechanisms in autophagy research.

How might ULK1 Antibody, HRP conjugated be utilized in studying novel ULK1-Recruiting Chimeras (ULKRECs)?

The development of ULK1-Recruiting Chimeras (ULKRECs) represents a cutting-edge approach for inducing targeted autophagy. These chimeric molecules combine an ULK1 agonist with a targeting ligand to direct ULK1 activity to specific cellular locations . ULK1 Antibody, HRP conjugated can be integrated into ULKREC research through several sophisticated approaches:

• Quantification of ULK1 Recruitment:
  ULKRECs are designed to recruit and activate ULK1 at specific cellular targets, such as mitochondria . Researchers can:

  • Develop ELISA-based assays to quantify ULK1 recruitment to specific cellular fractions

  • Compare ULK1 levels in mitochondrial fractions before and after ULKREC treatment

  • Establish dose-response relationships between ULKREC concentration and local ULK1 activity

• Validation of ULKREC Mechanism of Action:
  Understanding how ULKRECs induce targeted autophagy requires detailed mechanistic studies. Approaches include:

  • Measuring ULK1 activation status at targeted sites using phospho-specific antibodies

  • Correlating local ULK1 recruitment with downstream autophagy markers

  • Comparing the efficiency of different ULKREC designs in recruiting and activating ULK1

• Application in Disease Models:
  ULKRECs show promise for therapeutic development, particularly in diseases involving dysfunctional organelles . Research strategies include:

  • Quantifying ULK1 recruitment and activation in Parkinson's disease models

  • Measuring mitophagy induction in patient-derived fibroblasts

  • Evaluating the effectiveness of ULKRECs in bypassing defective PRKN/PINK pathways

• Comparative Analysis with Other Targeted Autophagy Approaches:
  ULKRECs represent an alternative to other approaches like PROTACs and conventional AUTACs. Experimental designs include:

  • Directly comparing ULK1 recruitment efficiency between different autophagy-inducing technologies

  • Assessing selectivity profiles using organelle-specific markers

  • Evaluating the kinetics of autophagic degradation induced by different approaches

This advanced application highlights how ULK1 Antibody, HRP conjugated can contribute to pioneering research in targeted autophagy.

What are common troubleshooting approaches for inconsistent results with ULK1 Antibody, HRP conjugated?

When encountering inconsistent results with ULK1 Antibody, HRP conjugated, researchers should consider the following methodological troubleshooting approaches:

• Antibody Storage and Handling Issues:

  • Avoid repeated freeze-thaw cycles that can degrade antibody activity

  • Store aliquoted antibody at recommended temperatures (typically -20°C for long-term)

  • Check for signs of precipitation or contamination before use

  • Verify HRP activity using direct substrate tests if activity loss is suspected

• Sample Preparation Considerations:

  • Ensure consistent protein extraction methods across experiments

  • Verify protein concentration using reliable quantification methods

  • Consider the phosphorylation state of ULK1, which varies with cell treatment conditions

  • Use phosphatase inhibitors in lysis buffers to preserve physiological phosphorylation status

• Assay-Specific Optimization:

  • Re-optimize antibody dilution through a systematic dilution series

  • Adjust incubation times and temperatures to enhance signal-to-noise ratio

  • Test different blocking reagents to reduce background

  • Consider switching detection substrates if HRP activity is suboptimal

• Biological Variability Factors:

  • Account for ULK1 expression changes under different cell culture conditions

  • Control for autophagy status, as ULK1 levels and localization change during autophagy

  • Consider cell density effects, as contact inhibition can alter autophagy pathways

  • Standardize treatment times when using autophagy modulators

Systematic documentation of these variables across experiments facilitates identification of sources of inconsistency.

How can researchers optimize ULK1 Antibody, HRP conjugated for use beyond ELISA applications?

While ULK1 Antibody, HRP conjugated is validated primarily for ELISA applications , researchers may adapt it for other techniques with careful optimization:

• Western Blotting Adaptation:

  • Use lower antibody concentrations (typically 1:2000-1:5000 dilution)

  • Optimize transfer conditions for high molecular weight proteins (ULK1 is ~112 kDa)

  • Employ direct detection using chemiluminescent HRP substrates

  • Consider longer exposure times but watch for background development

  • Implementation of a dot blot test series can help determine optimal concentration

• Immunocytochemistry Optimization:

  • Test fixation methods (paraformaldehyde vs. methanol) for optimal epitope preservation

  • Include permeabilization optimization (Triton X-100, saponin, or methanol concentrations)

  • Use higher antibody concentrations than ELISA (typically 1:100-1:500)

  • Extend incubation times (overnight at 4°C may improve sensitivity)

  • Employ tyramide signal amplification systems compatible with HRP

• Flow Cytometry Adaptation:

  • Optimize cell fixation and permeabilization for intracellular detection

  • Use compensation controls to account for HRP spectral properties

  • Consider using diluted antibody (1:200-1:500) to reduce non-specific binding

  • Establish clear positive and negative population controls

• Multiplexing Considerations:

  • When combining with other detection methods, ensure HRP detection is performed last

  • Use appropriate quenching steps when sequential staining is required

  • Consider stripping and reprobing protocols for sequential detection

  • Test for cross-reactivity with other detection systems

A systematic optimization approach with appropriate controls will maximize the utility of ULK1 Antibody, HRP conjugated beyond its primary ELISA application.

How might ULK1 Antibody, HRP conjugated contribute to research on ULK1's role in neurological disorders?

ULK1 plays important roles in neuronal differentiation and is required for granule cell axon formation . ULK1 Antibody, HRP conjugated can be incorporated into several emerging research approaches investigating neurological disorders:

• Neurodegenerative Disease Models:

  • Quantify ULK1 expression levels in Parkinson's disease models, where mitophagy defects are implicated

  • Monitor ULK1 activity in cellular models of neurodegeneration

  • Correlate ULK1 levels with mitochondrial clearance in PRKN/PINK1-deficient models

  • Develop high-throughput ELISA screening assays for compounds that modulate ULK1 activity in neuronal cells

• Axonal Development and Regeneration Studies:

  • Track ULK1 expression during different stages of neuronal differentiation

  • Compare ULK1 levels between developing and mature neurons

  • Investigate ULK1 expression changes following axonal injury

  • Correlate ULK1 activity with regenerative capacity in different neuronal populations

• Therapeutic Development for Neurological Disorders:

  • Screen for compounds that modulate ULK1 activity in neuronal contexts

  • Evaluate the effects of ULK1-targeting therapies on autophagy-related clearance of protein aggregates

  • Assess the potential of ULKRECs with neuronal targeting capabilities

  • Develop biomarker assays for monitoring ULK1 activity in cerebrospinal fluid

This emerging research direction highlights the potential of ULK1 as a therapeutic target in neurological disorders.

What is the potential role of ULK1 detection in developing combination cancer therapies?

Recent research has uncovered important connections between ULK1 inhibition and cancer immunotherapy, particularly in the context of combination treatments . ULK1 Antibody, HRP conjugated can contribute to this emerging field through:

• Biomarker Development for Immunotherapy Response:

  • The combination of ULK1 inhibition with PD-1 blockade has shown enhanced efficacy in tumor regression models

  • ULK1 Antibody, HRP conjugated can be used to develop ELISA-based biomarker assays that:

    • Predict response to combination therapy

    • Monitor treatment efficacy through serial sampling

    • Identify optimal treatment windows based on ULK1 activity levels

• Mechanistic Studies of ULK1 in Antigen Presentation:

  • ULK1 inhibition enhances immunoproteasome activity and antigen presentation

  • Research approaches include:

    • Quantifying changes in ULK1 expression during antigen processing

    • Correlating ULK1 levels with immunoproteasome component expression

    • Investigating the relationship between autophagy and antigen presentation machinery

• Personalized Treatment Approaches:

  • LKB1 mutation status affects response to ULK1 inhibition and immunotherapy

  • ULK1 detection can contribute to:

    • Stratifying patients based on tumor ULK1 expression patterns

    • Developing companion diagnostics for ULK1-targeting therapies

    • Monitoring acquired resistance mechanisms involving ULK1 pathway alterations