ADCK3 Antibody

What is ADCK3 and what are its primary functions in cellular metabolism?

ADCK3 functions as an atypical kinase involved in the biosynthesis of coenzyme Q (ubiquinone), which is an essential lipid-soluble electron transporter required for aerobic cellular respiration. Although initially classified as a protein kinase, recent research indicates ADCK3 likely does not function as a conventional protein kinase but rather may act as a small molecule kinase, possibly phosphorylating prenyl lipids in the ubiquinone biosynthesis pathway .

ADCK3 influences the modulation of mitochondrial complex components through phosphorylation, thereby stabilizing the function and efficiency of energy conversion processes within mitochondria . Interestingly, ADCK3 shows an unusual selectivity for binding ADP over ATP, which distinguishes it from typical kinases . ADCK3 is also known by several other names including CABC1, PP265, and COQ8A .

What types of ADCK3 antibodies are commercially available for research applications?

Based on the available information, there are multiple types of ADCK3 antibodies available for research applications:

• Polyclonal Antibodies:

  • Rabbit polyclonal antibodies, such as ab230897, are available with validated applications for Western blot (WB), immunohistochemistry on paraffin sections (IHC-P), and immunocytochemistry/immunofluorescence (ICC/IF) in human samples .

• Monoclonal Antibodies:

  • Mouse monoclonal antibodies, such as clone 5A4, are available with reactivity against human, mouse, and rat samples .

These antibodies differ in their host species, clonality, and validated applications, providing researchers with options based on their specific experimental needs.

What validated applications are supported by available ADCK3 antibodies?

ADCK3 antibodies have been validated for several key research applications:

For the rabbit polyclonal antibody (ab230897), Western blotting (WB), immunohistochemistry on paraffin sections (IHC-P), and immunocytochemistry/immunofluorescence (ICC/IF) have been validated for human samples . When choosing an antibody for your research, it's essential to consider whether the application has been directly tested or predicted to work based on sequence homology.

How should researchers validate ADCK3 antibodies before use in critical experiments?

Proper validation of ADCK3 antibodies is crucial for ensuring experimental reliability:

• Positive and Negative Controls:

  • Use samples with known ADCK3 expression levels (positive control)

  • Include ADCK3 knockout or knockdown samples as negative controls, similar to those generated using CRISPR-Cas9 as described in the literature

• Cross-Reactivity Testing:

  • Test antibody specificity across multiple species if working with non-human models

  • Verify lack of cross-reactivity with related proteins, particularly other ADCK family members

• Application-Specific Validation:

  • For Western blot: Confirm single band at expected molecular weight

  • For IHC/ICC: Validate subcellular localization (primarily mitochondrial for ADCK3)

  • For IP: Confirm pulldown of target protein by mass spectrometry

• Lot-to-Lot Consistency:

  • When possible, test antibodies from different lots to ensure reproducibility

Following these validation steps will maximize the reliability of results obtained using ADCK3 antibodies.

How is ADCK3 implicated in cancer biology, and how can antibodies help investigate these pathways?

Recent research has revealed ADCK3 as an important regulator in cancer biology, particularly in endometrial cancer (EC). ADCK3 has been identified as a key regulator of EC cells in response to medroxyprogesterone acetate (MPA) treatment . Specifically:

• Ferroptosis Regulation:

  • Loss of ADCK3 markedly alleviates MPA-induced cell death in EC cells

  • Mechanistically, ADCK3 deficiency suppresses MPA-mediated ferroptosis by preventing arachidonate 15-lipoxygenase (ALOX15) transcriptional activation

• p53 Pathway Interaction:

  • ADCK3 has been validated as a direct downstream target of the tumor suppressor p53 in EC cells

  • The p53-ADCK3 axis can be stimulated by small-molecule compounds like Nutlin3A, which synergizes with MPA to efficiently inhibit EC cell growth

ADCK3 antibodies are crucial tools for investigating these pathways through techniques such as:

• Western blotting to quantify ADCK3 protein levels after drug treatments

• Immunoprecipitation to identify interaction partners in the p53 pathway

• Immunocytochemistry to visualize subcellular localization during ferroptosis

• ChIP assays to study p53 binding to the ADCK3 promoter region

These applications enable researchers to unravel the complex roles of ADCK3 in cancer biology and potentially identify new therapeutic targets.

What methodologies are most effective for studying ADCK3 knockout models?

Creating and validating ADCK3 knockout models requires careful methodology. Based on published research, the following approach has proven effective:

• CRISPR-Cas9 Knockout Generation:

  • Design sgRNAs targeting ADCK3 exons (preferably early exons)

  • Clone sgRNA oligos into lentiCRISPR v2 construct (Addgene, 52961)

  • Generate lentivirus by transfecting HEK293T cells with the expression construct and packaging constructs (pMD2.G and psPAX2)

  • Infect target cells (e.g., Ishikawa cells for EC research) and select with puromycin

• Knockout Validation:

  • Western blot analysis to confirm protein depletion

  • Genomic DNA sequencing to verify mutations at the target site

  • Select at least two independent clones for experiments to control for off-target effects

• Functional Validation:

  • Assess mitochondrial function (oxygen consumption rate, ATP production)

  • Measure coenzyme Q levels to confirm metabolic impact

  • Evaluate cellular responses to stressors (e.g., MPA treatment in EC cells)

For gene expression analysis in ADCK3-KO models, RNA-seq followed by ssGSEA (single-sample Gene Set Enrichment Analysis) has been successfully applied to identify pathway alterations .

How can structure-based virtual screening be applied to identify ADCK3 inhibitors?

Structure-based virtual screening has proven effective for identifying novel ADCK3 inhibitors. A comprehensive methodology involves:

• Protein Structure Preparation and Pharmacophore Modeling:

  • Obtain the 3D structure of ADCK3 (PDB code: 5I35), which is complexed with AMPPNP (an ATP mimetic)

  • Remove non-structural water molecules, add hydrogens, and optimize conformation using tools like Mgtools 1.5.6

  • Generate a pharmacophore model using programs like MOE based on the co-crystallized ligand

• Key Pharmacophore Features for ADCK3 Inhibitors:

  • H-bond donor (located at the -NH₂ group of the adenine core)

  • H-bond acceptor (from the adenine ring nitrogen, interacting with backbone -NH of Val448)

  • Aromatic ring (located in the adenine core participating in T-shaped π-interactions)

  • These three features capture the crucial interactions for ADCK3 inhibition

• Virtual Screening Workflow:

  • Apply the three-point pharmacophore to screen diverse chemical libraries

  • Score compounds using molecular docking

  • Select top-ranking compounds for experimental validation

  • In a published study, screening ~170,000 compounds yielded 129 hits with inhibitory activity (16.1% hit rate)

• Validation through Biochemical Assays:

  • Test candidate compounds in dose-response biochemical assays

  • Incorporate counter-screening against related kinases (e.g., p38) to assess selectivity

  • The most potent compounds identified through this approach showed nanomolar to single-digit micromolar potency

This workflow represents a promising strategy for accelerated drug discovery targeting ADCK3 and can be extended to other targets.

What computational methods best predict ADCK3 inhibitor binding and efficacy?

Molecular dynamics (MD) simulations with metadynamics analysis provide the most comprehensive computational insights into ADCK3 inhibitor binding:

• MD Simulation Protocol:

  • Perform simulations at physiologically relevant temperature (310 K)

  • Use established force fields like CHARMM 27

  • Define an appropriate unit cell size (e.g., 80 × 80 × 80 ų) to encompass the entire ADCK3 protein

  • Simulate in the NVT ensemble with the SPC/E water model and 0.9% NaCl concentration

  • Run simulations for sufficient time (≥200 ns) to reach convergence

• Key Analyses to Perform:

  • Root-mean-square deviation (RMSD) to assess structural stability

  • Root-mean-square fluctuation (RMSF) to identify flexible regions

  • Hydrogen bond analysis to characterize protein-ligand interactions

  • Free energy calculations through metadynamics to estimate binding affinity

• Critical Findings from MD Studies:

  • ADCK3 inhibitors are predominantly noncovalent binders

  • van der Waals (vdW) and electrostatic interactions play dominant roles in protein-inhibitor binding

  • Binding free energies correlate with experimentally observed inhibitory activities, though not in strict linear fashion

• Limitations to Consider:

  • Accuracy may be influenced by simulation time and selected parameters

  • Steric hindrance can affect prediction quality

  • Conformational flexibility of inhibitors impacts binding activity

These computational approaches provide valuable guidance for medicinal chemistry optimization of ADCK3 inhibitors, particularly when combined with experimental validation.

What role does ADCK3 play in ferroptosis, and how can this be experimentally investigated?

ADCK3 has recently been identified as a key regulator of ferroptosis, particularly in the context of endometrial cancer response to MPA treatment:

• Mechanism of ADCK3 in Ferroptosis:

  • ADCK3 regulates ferroptosis through the transcriptional activation of arachidonate 15-lipoxygenase (ALOX15)

  • Loss of ADCK3 suppresses MPA-mediated ferroptosis by preventing ALOX15 activation

  • ADCK3 functions as a downstream target of p53, creating a p53-ADCK3-ALOX15 axis in ferroptosis regulation

• Experimental Approaches to Study This Pathway:

  a) Genetic Manipulation:

  • Generate ADCK3 knockout or knockdown models using CRISPR-Cas9 or shRNA

  • Create rescue experiments with wild-type or mutant ADCK3 to identify critical domains

  b) Ferroptosis Assays:

  • Measure lipid peroxidation using C11-BODIPY or MDA assays

  • Assess glutathione depletion and GPX4 activity

  • Determine cell death using specific ferroptosis inhibitors (ferrostatin-1, liproxstatin-1) as controls

  c) Transcriptional Analysis:

  • Perform ChIP assays to investigate p53 binding to ADCK3 regulatory regions

  • Use RT-qPCR and Western blot to measure ALOX15 expression in response to ADCK3 manipulation

  • Apply RNA-seq to identify global transcriptional changes

• Combined Therapeutic Approaches:

  • Investigate synergistic effects between p53 activators (e.g., Nutlin3A) and MPA

  • Measure cell growth inhibition and ferroptotic markers

  • Assess ADCK3 and ALOX15 expression levels following combination treatment

This emerging area of research highlights ADCK3 as a potential therapeutic target in cancers where ferroptosis induction could be beneficial.

What are common technical challenges when working with ADCK3 antibodies?

Researchers may encounter several technical challenges when working with ADCK3 antibodies:

• Specificity Issues:

  • Cross-reactivity with other ADCK family members (ADCK1-5)

  • Non-specific binding in certain tissues or cell types

  Solution: Validate antibody specificity using ADCK3 knockout controls and test multiple antibodies from different sources if possible.

• Mitochondrial Localization Challenges:

  • ADCK3's mitochondrial localization can complicate immunostaining

  • May require specialized permeabilization for adequate antibody access

  Solution: Use optimized fixation and permeabilization protocols; consider co-staining with established mitochondrial markers.

• Post-translational Modifications:

  • Potential epitope masking due to phosphorylation or other modifications

  • May affect antibody recognition in different physiological states

  Solution: Use multiple antibodies targeting different epitopes; consider phospho-specific antibodies if studying ADCK3 regulation.

• Low Expression Levels:

  • ADCK3 may be expressed at relatively low levels in some cell types

  • May require sensitive detection methods

  Solution: Optimize protein loading for Western blot; consider signal amplification methods for immunostaining; use appropriate positive controls.

How can researchers optimize Western blot protocols for ADCK3 detection?

Optimizing Western blot protocols for ADCK3 detection requires attention to several key parameters:

• Sample Preparation:

  • Use RIPA or NP-40 buffer with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylation

  • For mitochondrial enrichment: consider subcellular fractionation protocols

• Gel Electrophoresis and Transfer:

  • 10-12% SDS-PAGE gels typically work well for ADCK3 (~70 kDa)

  • Transfer conditions: 100V for 60-90 minutes or 30V overnight at 4°C

  • Consider wet transfer for more efficient transfer of larger proteins

• Blocking and Antibody Incubation:

  • 5% non-fat dry milk or BSA in TBST

  • Primary antibody dilution: start with manufacturer's recommendation (typically 1:1000 for rabbit polyclonal)

  • Incubate at 4°C overnight for optimal signal-to-noise ratio

• Detection Optimization:

  • Use high-sensitivity ECL substrates for low-abundance targets

  • Consider fluorescent secondary antibodies for more quantitative analysis

  • Optimize exposure times to avoid saturation

• Troubleshooting Common Issues:

  • High background: increase washing steps, optimize antibody dilution

  • Weak signal: increase protein loading, extend exposure time, use signal enhancement systems

  • Multiple bands: verify specificity with ADCK3 knockout control, adjust antibody concentration

By systematically optimizing these parameters, researchers can achieve reliable and reproducible detection of ADCK3 protein by Western blot.

How can ADCK3 antibodies contribute to studying mitochondrial disorders?

ADCK3 antibodies are valuable tools for investigating mitochondrial disorders, particularly those related to coenzyme Q deficiency:

• Diagnostic Applications:

  • Assess ADCK3 protein levels in patient samples

  • Determine subcellular localization changes in disease states

  • Evaluate post-translational modifications that may be altered in pathological conditions

• Research Applications:

  • Immunoprecipitation to identify novel ADCK3 interaction partners

  • ChIP-seq to map genomic binding sites of transcription factors regulating ADCK3

  • Proximity labeling (BioID, APEX) combined with ADCK3 antibodies to map the local protein environment

• Therapeutic Development Support:

  • Monitor ADCK3 expression in response to candidate therapeutics

  • Assess restoration of normal ADCK3 function in rescue experiments

  • Evaluate target engagement of ADCK3-directed compounds

• Emerging Applications:

  • Super-resolution microscopy to precisely localize ADCK3 within mitochondrial subcompartments

  • Single-cell proteomics to assess ADCK3 expression heterogeneity

  • Mass cytometry (CyTOF) incorporating ADCK3 antibodies for multiparametric analysis of patient samples

These applications position ADCK3 antibodies as essential tools for advancing our understanding of mitochondrial disorders and developing targeted therapeutic approaches.

What are promising future directions for ADCK3 research in cancer biology?

Based on recent discoveries, several promising research directions emerge for ADCK3 in cancer biology:

• Therapeutic Targeting of the p53-ADCK3-ALOX15 Axis:

  • Development of small molecules that modulate ADCK3 activity

  • Combination therapies activating p53 and targeting ADCK3-dependent pathways

  • Exploration of synthetic lethal interactions with ADCK3 inhibition

• Biomarker Development:

  • Evaluation of ADCK3 expression as a predictive biomarker for MPA response in endometrial cancer

  • Correlation of ADCK3 levels with ferroptosis sensitivity across cancer types

  • Development of companion diagnostics for ADCK3-targeted therapies

• Metabolic Vulnerabilities:

  • Investigation of how ADCK3 links mitochondrial function to ferroptosis sensitivity

  • Exploration of ADCK3's role in cancer metabolism beyond ferroptosis

  • Identification of metabolic dependencies created by ADCK3 alterations

• Resistance Mechanisms:

  • Understanding how cancer cells develop resistance to ADCK3-dependent cell death

  • Mapping compensatory pathways activated upon ADCK3 inhibition

  • Development of strategies to overcome resistance to ADCK3-targeted therapies