AK2 Antibody

What is AK2 and why is it important in cellular metabolism?

AK2 (Adenylate kinase 2, mitochondrial) is an enzyme that catalyzes the reversible transfer of the terminal phosphate group between ATP and AMP. It plays a critical role in cellular energy homeostasis and in adenine nucleotide metabolism . This 26 kDa protein is primarily localized in mitochondria, where it facilitates the reversible conversion of ADP into ATP and AMP playing a critical role in cellular energy homeostasis . Its expression allows for efficient energy transfer essential for normal mitochondrial function and cellular metabolism . Adenylate kinase activity is critical for regulation of phosphate utilization and the AMP de novo biosynthesis pathways . Additionally, research has established that AK2 plays a key role in hematopoiesis, with studies showing that disturbed adenine nucleotide metabolism through AK2 knockdown or mutation can lead to increased reactive oxygen species (ROS) production .

What types of AK2 antibodies are available for research applications?

Several types of AK2 antibodies are available for research applications, each with specific characteristics and validated applications. The main types include:

• Polyclonal AK2 antibodies: Such as the rabbit polyclonal antibody (11014-1-AP) from Proteintech, which has been validated for Western blot, immunohistochemistry, immunofluorescence, immunoprecipitation, and ELISA applications . This antibody shows reactivity with human, mouse, and rat samples .

• Monoclonal AK2 antibodies: These include rabbit recombinant monoclonal antibodies like Abcam's [EPR11387(B)] (ab157206) and [EPR11388(B)] (ab166901) . The EPR11388(B) antibody has been validated for IHC-P, IP, WB, and ICC/IF applications with reactivity to human, mouse, and rat samples . Additionally, mouse anti-human monoclonal antibodies are available, such as the one mentioned in search result .

These antibodies differ in their host species, clonality, validated applications, and reactivity with target species. For instance, the rabbit polyclonal antibody from Proteintech has been positively validated for detecting AK2 in various cell lines including HL-60, HeLa, and HepG2 cells, as well as in mouse kidney, mouse liver, rat kidney, and rat liver tissues . The choice between these antibodies depends on the specific research application, target species, and experimental design requirements.

How does AK2 antibody selection impact experimental outcomes?

The selection of an appropriate AK2 antibody can significantly impact experimental outcomes, particularly regarding specificity, sensitivity, and application compatibility. When selecting an AK2 antibody, researchers should consider:

• Antibody specificity: Monoclonal antibodies like Abcam's [EPR11387(B)] and [EPR11388(B)] typically offer higher specificity for particular epitopes, while polyclonal antibodies such as Proteintech's 11014-1-AP may recognize multiple epitopes, potentially providing stronger signals but with increased risk of cross-reactivity . For crucial experiments, validation using AK2 knockout controls is essential, as demonstrated in the search results showing antibody validation using Human AK2 knockout HEK-293T cell lines .

• Application compatibility: Different antibodies perform optimally in specific applications. For example, Proteintech's AK2 antibody (11014-1-AP) is recommended at dilutions of 1:1000-1:4000 for Western blot, 1:50-1:500 for IHC, and 1:50-1:500 for IF/ICC applications . The Abcam antibody [EPR11388(B)] has been specifically validated for IHC-P, IP, WB, and ICC/IF applications .

• Species reactivity: Experimental outcomes can be compromised if the antibody doesn't recognize the species being studied. The search results indicate that the available AK2 antibodies show reactivity with human, mouse, and rat samples, with some citations also mentioning zebrafish reactivity .

• Antibody format and conjugation: Most available AK2 antibodies are unconjugated, such as the Proteintech antibody which is supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . This format allows flexibility for various detection methods but requires appropriate secondary antibodies for visualization.

What are the optimal conditions for Western blot detection of AK2?

For optimal Western blot detection of AK2, researchers should consider the following methodological approach:

• Sample preparation: AK2 is expressed in various cell types and tissues. According to the search results, positive Western blot detection has been achieved in HL-60 cells, HeLa cells, HepG2 cells, mouse kidney tissue, mouse liver tissue, rat kidney tissue, and rat liver tissue . Proper lysis buffers that preserve protein integrity while effectively extracting AK2 should be employed.

• Protein amount and loading: Since AK2 has a calculated and observed molecular weight of 26 kDa, appropriate molecular weight markers should be used . Standard protein loading amounts (20-50 μg of total protein) are typically sufficient for detection.

• Antibody selection and dilution: For the Proteintech polyclonal antibody (11014-1-AP), the recommended dilution range is 1:1000-1:4000 for Western blot applications . The Abcam monoclonal antibodies should be used according to their specific recommended dilutions.

• Controls: For definitive validation, the search results demonstrate the use of AK2 knockout cell lines (Human AK2 knockout HEK-293T cell line) as negative controls . Additionally, appropriate loading controls such as GAPDH or α-Tubulin should be included as shown in the validation data from Abcam .

• Detection system: Standard chemiluminescent or fluorescent secondary antibody detection systems compatible with the primary antibody host species (rabbit for the antibodies mentioned in the search results) are appropriate.

• Expected results: AK2 should be detected as a single band at approximately 26 kDa . Multiple or off-target bands may indicate non-specific binding or experimental issues.

• Troubleshooting: In case of weak signal, consider increasing the antibody concentration, extending incubation time, or improving transfer efficiency. For high background, increase washing steps or optimize blocking conditions.

How should I optimize immunohistochemistry protocols for AK2 detection in tissue samples?

For optimal immunohistochemistry detection of AK2 in tissue samples, consider the following methodology:

• Tissue preparation and antigen retrieval: The Proteintech antibody (11014-1-AP) has been positively validated for IHC in human pancreas cancer tissue . For optimal results, perform antigen retrieval with TE buffer pH 9.0, although citrate buffer pH 6.0 can serve as an alternative . Proper fixation and embedding techniques are crucial for preserving both tissue morphology and antigen integrity.

• Blocking and antibody incubation: Use appropriate blocking solutions to minimize non-specific binding. The Proteintech antibody should be used at 1:50-1:500 dilution for IHC . For the Abcam antibodies [EPR11387(B)] and [EPR11388(B)], follow their specific recommended dilutions for IHC-P applications . Optimize incubation time and temperature (typically overnight at 4°C or 1-2 hours at room temperature).

• Detection systems: Use a detection system compatible with the primary antibody host species (rabbit for the antibodies mentioned in the search results). Chromogenic detection systems (DAB or AEC) are commonly employed for IHC applications, with hematoxylin counterstaining for visualizing tissue architecture.

• Controls: Include positive control tissues known to express AK2 (based on the search results, pancreatic tissue would be appropriate) . Negative controls should include omission of primary antibody or, ideally, tissues from knockout models if available.

• Visualization and analysis: AK2 is primarily localized in mitochondria, so expect a cytoplasmic pattern with potential mitochondrial emphasis. Compare expression levels across different cell types within the tissue to identify cell-specific expression patterns.

• Quantification: For quantitative analysis, consider using digital image analysis software to measure staining intensity and distribution while accounting for background and non-specific staining.

What are the key considerations for immunofluorescence detection of AK2 in cell culture?

For effective immunofluorescence detection of AK2 in cell culture, researchers should consider the following methodological approach:

• Cell selection and preparation: According to the search results, HepG2 cells have been successfully used for AK2 immunofluorescence studies . Grow cells on appropriate coverslips or chamber slides under standard culture conditions. Fixation with 4% paraformaldehyde followed by permeabilization with Triton X-100 or methanol is typically suitable for intracellular proteins like AK2.

• Antibody selection and dilution: The Proteintech antibody (11014-1-AP) should be used at 1:50-1:500 dilution for IF/ICC applications . For the Abcam antibody [EPR11388(B)] (ab166901), follow their specific recommendations for ICC/IF applications . Incubation conditions typically involve 1-2 hours at room temperature or overnight at 4°C in a humidified chamber.

• Co-staining considerations: Since AK2 is a mitochondrial protein, consider co-staining with established mitochondrial markers to confirm proper localization. Nuclear counterstaining with DAPI or Hoechst is recommended for proper cellular orientation.

• Controls: Include appropriate negative controls (primary antibody omission, non-specific IgG) and, if available, cells with modified AK2 expression (knockdown, knockout, or overexpression) as biological controls.

• Image acquisition: Confocal microscopy is preferred for optimal visualization of mitochondrial localization. Capture Z-stack images to ensure complete visualization of the three-dimensional cellular structure. Use consistent exposure settings when comparing different experimental conditions.

• Analysis: Quantify AK2 expression using appropriate image analysis software. Consider colocalization analysis with mitochondrial markers to confirm proper localization. For comparative studies, ensure standardized acquisition parameters and analysis methods.

• Troubleshooting: For weak signal, optimize antibody concentration, incubation time, or permeabilization conditions. For high background, increase washing steps or modify blocking conditions.

How can I design experiments to investigate AK2's role in cellular energy homeostasis?

To investigate AK2's role in cellular energy homeostasis, researchers should consider the following comprehensive experimental design approach:

By integrating these experimental approaches, researchers can comprehensively characterize AK2's contributions to cellular energy homeostasis and identify its role in normal physiology and pathological conditions.

What approaches can be used to study AK2's involvement in oxidative stress pathways?

Based on search result , which indicates AK2's potential involvement in oxidative stress regulation, researchers can implement the following methodological approaches:

• Experimental models for studying AK2-oxidative stress relationships:

  • Cell culture systems: T cells and PBMCs are mentioned in search result as relevant models for studying AK2's role in oxidative stress responses . Establish additional model systems with AK2 knockdown, knockout, or overexpression to directly assess AK2's impact on ROS regulation.

  • Stress induction protocols: Employ radiation exposure (e.g., 8 Gy as used in the referenced study), chemical oxidants (H₂O₂, paraquat), or metabolic stress conditions to provoke oxidative stress responses .

• ROS detection and quantification methodologies:

  • Utilize fluorescent probes specific for different ROS species (H₂DCFDA for general ROS, MitoSOX for mitochondrial superoxide)

  • Implement flow cytometry for quantitative single-cell analysis of ROS levels

  • Measure oxidized and reduced glutathione ratios (GSH/GSSG) as indicators of cellular redox state

  • Assess lipid peroxidation products (MDA, 4-HNE) and protein oxidation markers (protein carbonyls)

• Molecular pathway analysis:

  • Investigate connections between AK2 and NOX4, which was measured in PBMCs after irradiation according to search result

  • Examine how AK2 expression levels affect antioxidant enzyme activities (SOD, catalase, glutathione peroxidase)

  • Analyze activation of redox-sensitive transcription factors (Nrf2, NF-κB) in response to AK2 modulation

  • Assess mitochondrial function parameters in relation to AK2 expression and oxidative stress

• Translational research approaches:

  • Correlate AK2 expression with oxidative stress markers in patient samples

  • Develop predictive biomarkers based on AK2 expression for radiation sensitivity, as suggested by search result

  • Investigate protective interventions targeting AK2-related pathways against oxidative damage

  • Explore connections between AK2, oxidative stress, and specific disease states

By systematically applying these methodological approaches, researchers can comprehensively characterize AK2's specific contributions to oxidative stress regulation and identify potential therapeutic targets for conditions associated with oxidative damage.

How can AK2 antibodies be used to investigate protein-protein interactions and complex formation?

AK2 antibodies can be powerful tools for investigating protein-protein interactions and complex formation through the following methodological approaches:

• Co-immunoprecipitation (Co-IP) strategies:

  • Use AK2 antibodies for immunoprecipitation followed by Western blotting for potential interacting partners. According to search result , the Proteintech AK2 antibody (11014-1-AP) is recommended at 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate for IP applications and has been validated in mouse liver tissue . Similarly, the Abcam antibody [EPR11388(B)] (ab166901) is validated for IP applications .

  • Perform reciprocal Co-IP experiments (immunoprecipitate with antibodies against suspected interacting partners and blot for AK2)

  • Consider using crosslinking approaches to capture transient or weak interactions

  • Implement stringent controls including isotype-matched non-specific antibodies and AK2 knockout samples

• Advanced protein interaction analysis techniques:

  • Proximity ligation assay (PLA) to visualize protein-protein interactions in situ with high sensitivity

  • FRET (Förster Resonance Energy Transfer) or BRET (Bioluminescence Resonance Energy Transfer) to study interactions in living cells

  • Mass spectrometry analysis of immunoprecipitated complexes to identify novel interacting partners

  • Yeast two-hybrid or mammalian two-hybrid screening using AK2 as bait to identify potential interactors

• Subcellular localization and complex visualization:

  • Use immunofluorescence with AK2 antibodies together with markers for potential interacting partners

  • Perform subcellular fractionation followed by immunoblotting to determine compartment-specific interactions

  • Apply super-resolution microscopy techniques to visualize protein complexes at nanoscale resolution

  • Conduct time-lapse imaging to track dynamic complex formation under various cellular conditions

• Functional validation of interactions:

  • Design mutation studies targeting predicted interaction domains

  • Perform competition assays with peptides or small molecules targeting interaction sites

  • Assess functional consequences of disrupting specific interactions

  • Correlate interaction patterns with functional readouts (enzyme activity, cellular phenotypes)

• Context-dependent interaction studies:

  • Investigate how AK2 interactions change under different metabolic states

  • Examine interaction patterns following exposure to stressors (such as radiation, based on search result )

  • Compare interaction networks across different cell types or tissues

  • Assess how disease-associated mutations affect AK2's interactome

By implementing these methodological approaches, researchers can comprehensively map AK2's protein interaction network and understand how these interactions contribute to its functions in cellular energy homeostasis, hematopoiesis, and stress responses.

How can I troubleshoot common issues with AK2 detection in Western blotting?

When encountering challenges with AK2 detection in Western blotting, consider the following systematic troubleshooting approaches:

• No signal or weak signal:

  • Antibody concentration: If using the Proteintech antibody (11014-1-AP), try increasing concentration within the recommended 1:1000-1:4000 dilution range . Start with 1:1000 and adjust as needed.

  • Protein loading: Ensure sufficient protein loading (30-50 μg of total protein). AK2 has a molecular weight of 26 kDa, so verify that your gel percentage is appropriate for this size range .

  • Transfer efficiency: Check transfer by staining membrane with Ponceau S before antibody incubation. Optimize transfer conditions for proteins in the 26 kDa range.

  • Exposure time: For chemiluminescent detection, try longer exposure times. For fluorescent detection, adjust scanner settings.

  • Sample preparation: Ensure complete protein denaturation and use fresh reducing agents in loading buffer.

• Multiple bands or high background:

  • Antibody specificity: Validate using AK2 knockout samples if available. The search results mention Human AK2 knockout HEK-293T cell line as a useful negative control .

  • Blocking: Optimize blocking conditions (time, temperature, and blocking agent concentration).

  • Washing: Increase number and duration of washing steps with TBST/PBST.

  • Antibody dilution: If background is high, try more dilute antibody concentrations.

• Inconsistent results between experiments:

  • Sample handling: Standardize protein extraction and quantification methods.

  • Antibody storage: Follow the recommended storage conditions. The Proteintech antibody should be stored at -20°C and is stable for one year after shipment .

  • Loading controls: Always include appropriate loading controls (GAPDH, β-actin, or α-Tubulin).

  • Gel conditions: Maintain consistent gel percentage, running conditions, and transfer parameters.

• Cell/tissue-specific issues:

  • Expression levels: According to search results, AK2 has been successfully detected in HL-60 cells, HeLa cells, HepG2 cells, mouse kidney tissue, mouse liver tissue, rat kidney tissue, and rat liver tissue . If working with other cell types, expression levels may vary.

  • Extraction protocol: Optimize lysis buffers for specific tissue types, particularly for tissues with high protease activity.

  • Species reactivity: Ensure the selected antibody reacts with your species of interest. The search results indicate human, mouse, and rat reactivity for the discussed antibodies .

By systematically addressing these potential issues, researchers can optimize Western blotting protocols for reliable AK2 detection across different experimental systems.

What approaches can validate AK2 antibody specificity in different experimental systems?

Validating antibody specificity is crucial for ensuring reliable experimental results. Here are methodological approaches for validating AK2 antibody specificity across different applications:

• Genetic validation strategies:

  • Knockout controls: The search results indicate that Human AK2 knockout HEK-293T cell lines have been used to validate antibody specificity . Using such knockout controls provides definitive evidence of antibody specificity.

  • Knockdown validation: Compare signals between AK2 siRNA/shRNA-treated cells and control cells to confirm signal reduction correlating with decreased target expression.

  • Overexpression validation: Test antibody on cells overexpressing tagged AK2 to confirm signal increase.

• Application-specific validation approaches:

  a. For Western blotting:

  • Verify single band detection at the expected molecular weight (26 kDa for AK2)

  • Perform peptide competition assays to block specific binding

  • Compare multiple antibodies targeting different epitopes of AK2

  • Include positive control tissues known to express AK2 (search results indicate liver and kidney tissues)

  b. For Immunohistochemistry/Immunofluorescence:

  • Compare staining patterns with known subcellular localization (mitochondrial for AK2)

  • Perform co-localization studies with established mitochondrial markers

  • Validate specificity using tissues or cells with genetically modified AK2 expression

  • Ensure absence of signal in negative control conditions (primary antibody omission, isotype controls)

  c. For Immunoprecipitation:

  • Validate IP efficiency by Western blotting a portion of immunoprecipitated material

  • Use mass spectrometry to confirm identity of immunoprecipitated proteins

  • Perform reciprocal co-IP experiments when studying protein-protein interactions

• Cross-validation approaches:

  • Compare protein detection results with mRNA expression data (though search result indicates protein stability rather than transcriptional effects may be important for AK2)

  • Validate across multiple antibodies from different vendors or clones

  • Use orthogonal methods to confirm findings (e.g., mass spectrometry validation of Western blot results)

• Documentation and reporting practices:

  • Document all validation experiments with detailed methods and results

  • Include validation data when publishing research using the antibody

  • Report batch/lot numbers as antibody performance can vary between production lots

These systematic validation approaches ensure that experimental results with AK2 antibodies are reliable and reproducible across different research applications and experimental systems.

How can I optimize immunoprecipitation protocols using AK2 antibodies for studying interacting proteins?

Based on the search results indicating that AK2 antibodies have been validated for immunoprecipitation, here's a methodological approach to optimize IP protocols for studying AK2 protein interactions:

• Sample preparation considerations:

  • According to search result , the Proteintech AK2 antibody (11014-1-AP) has been validated for IP in mouse liver tissue . For other tissues or cell lines, optimization may be necessary.

  • Use gentle lysis buffers that preserve protein-protein interactions (e.g., NP-40, CHAPS, or Triton X-100 based buffers at 0.5-1% concentration).

  • Include protease inhibitors, phosphatase inhibitors, and EDTA to prevent degradation and maintain protein modifications.

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding before adding the specific antibody.

• Antibody selection and amount optimization:

  • For the Proteintech antibody (11014-1-AP), use 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate as recommended .

  • For the Abcam antibody [EPR11388(B)] (ab166901), follow their specific recommendations for IP applications .

  • Titrate antibody amounts to determine the optimal concentration for your specific sample type.

• Immunoprecipitation conditions optimization:

  • Test different antibody-to-lysate ratios (start with recommendations and adjust based on results).

  • Compare incubation times (2-4 hours vs. overnight) and temperatures (4°C is standard).

  • Evaluate different bead types (protein A, protein G, or protein A/G mix) based on the antibody isotype.

  • For rabbit antibodies like those in the search results, protein A beads often work well.

• Washing protocol optimization:

  • Balance washing stringency: too stringent may disrupt specific interactions; too gentle may retain non-specific binding.

  • Test different salt concentrations in wash buffers (150-500 mM NaCl).

  • Include detergents (0.1% Triton X-100 or NP-40) in wash buffers to reduce background.

  • Perform multiple washes (4-5 times) with fresh buffer each time.

• Critical controls to include:

  • Input control: Save 5-10% of pre-IP lysate to confirm target protein presence.

  • IgG control: Perform parallel IP with isotype-matched non-specific antibody.

  • Knockout/knockdown control: Where available, include samples with reduced AK2 expression.

  • Positive control: Include a known AK2-interacting protein in your analysis.

• Detection strategies for interacting proteins:

  • Western blot using antibodies against suspected interacting partners.

  • Mass spectrometry analysis for unbiased identification of co-immunoprecipitated proteins.

  • Targeted analysis for post-translational modifications on immunoprecipitated AK2.

  • Functional assays to validate biological relevance of identified interactions.

By implementing these optimization strategies, researchers can develop robust immunoprecipitation protocols using AK2 antibodies to reliably identify and characterize protein interaction networks in various experimental systems.

How is AK2 being investigated as a potential biomarker in radiation response studies?

Based on search result , AK2 has emerged as a promising biomarker candidate in radiation response studies. The methodological approaches for investigating this application include:

• Proteomic identification and validation strategies:

  • The referenced study used iTRAQ-based proteomic analysis to identify AK2 as differentially expressed in irradiated T lymphocytes from patients with varying degrees of radiation toxicity (grade ≥ 2 bf+ versus grade < 2 bf+) .

  • This discovery was validated using Western blotting to confirm differential AK2 protein expression between patient groups .

  • qRT-PCR analysis revealed comparable AK2 mRNA levels between groups, suggesting that protein stability rather than transcriptional regulation may be important in radiation response .

• Experimental models and radiation parameters:

  • T lymphocytes served as the primary experimental model for proteomic analysis identifying AK2 .

  • Peripheral blood mononuclear cells (PBMCs) were used to measure NOX4 levels after irradiation .

  • The study employed 8 Gy radiation doses to identify differential protein expression patterns .

  • The research focused on comparing patients with different grades of radiation toxicity (grade ≥ 2 bf+ versus grade < 2 bf+) .

• Mechanistic connections with oxidative stress pathways:

  • AK2 was identified among 23 proteins mainly involved in oxidation-reduction processes and RNA processing that were differentially expressed after radiation exposure .

  • The study noted that disturbed adenine nucleotide metabolism, through AK2 knockdown or mutation, can lead to increased reactive oxygen species (ROS) levels .

  • Oxidative stress was identified as a key biochemical event during radiation exposure and is often considered the main mediator of ionizing radiation's deleterious effects .

  • The research suggested that persistent oxidative changes, which may continue for days or months after initial radiation exposure, could explain long-term side effects observed after radiotherapy .

• Potential clinical applications:

  • AK2 could serve as a predictive biomarker for radiation sensitivity or toxicity risk.

  • The protein might be useful for patient stratification in radiotherapy planning.

  • Monitoring AK2 levels might help assess radiation damage or recovery.

  • Understanding AK2's role in radiation response could lead to protective interventions to reduce radiation toxicity.

This research direction highlights AK2's potential beyond its basic metabolic functions, positioning it as a candidate biomarker in radiation oncology with possible implications for personalized treatment approaches.

What role does AK2 play in mitochondrial function and how can this be studied?

AK2 is localized in the mitochondrial intermembrane space and plays critical roles in mitochondrial function. Here's a methodological framework for investigating these roles:

• Functional significance of AK2 in mitochondria:

  • AK2 catalyzes the reversible transfer of phosphate groups between ATP and AMP, playing an important role in cellular energy homeostasis and adenine nucleotide metabolism .

  • It contributes to energy transfer networks by facilitating the conversion of ADP to ATP and AMP, which is essential for normal mitochondrial function and cellular metabolism .

  • Adenylate kinase activity is critical for regulation of phosphate utilization and AMP de novo biosynthesis pathways .

  • AK2 may influence mitochondrial oxidative phosphorylation and ROS production, as studies have shown that AK2 knockdown or mutation can result in increased ROS levels .

• Experimental approaches to study AK2's mitochondrial functions:

  a. Mitochondrial isolation and functional analysis:

  • Isolate intact mitochondria from cells with normal, reduced, or increased AK2 expression

  • Measure mitochondrial respiration (oxygen consumption rate) using respirometry

  • Assess mitochondrial membrane potential using fluorescent dyes

  • Determine ATP production capacity in isolated mitochondria

  b. Live-cell mitochondrial dynamics:

  • Use fluorescent AK2 fusion proteins to track localization and dynamics

  • Implement time-lapse imaging to observe mitochondrial morphology changes

  • Apply photobleaching techniques (FRAP) to assess AK2 mobility within mitochondria

  • Employ mitochondrial-targeted sensors to measure local ATP/ADP ratios

  c. Mitochondrial stress responses:

  • Challenge cells with mitochondrial stressors (electron transport chain inhibitors, uncouplers)

  • Assess the impact of AK2 expression levels on mitochondrial stress tolerance

  • Measure mitochondrial ROS production using specific probes

  • Analyze mitochondrial quality control mechanisms (mitophagy, biogenesis)

• AK2 interaction with mitochondrial proteins:

  • Use mitochondrial sub-fractionation to precisely localize AK2

  • Perform proximity labeling techniques to identify nearby proteins

  • Conduct co-immunoprecipitation with AK2 antibodies to identify mitochondrial protein partners

  • Investigate interactions with components of oxidative phosphorylation complexes

• Disease-relevant mitochondrial dysfunction:

  • Analyze AK2 expression and function in mitochondrial disease models

  • Study AK2's role in conditions with known mitochondrial involvement

  • Investigate connections between AK2, mitochondrial function, and oxidative stress in radiation response

  • Examine AK2's contribution to mitochondrial-dependent processes in hematopoiesis

By applying these methodological approaches, researchers can comprehensively characterize AK2's specific contributions to mitochondrial function and identify potential therapeutic targets for conditions associated with mitochondrial dysfunction.

How can high-throughput approaches be used to explore AK2 function in different cellular contexts?

High-throughput approaches offer powerful tools for comprehensively exploring AK2 function across diverse cellular contexts. Here are methodological strategies for implementing such approaches:

• Genomic and transcriptomic screening strategies:

  • CRISPR-Cas9 screens to identify genes that synthetically interact with AK2

  • RNAi screens to identify modulators of AK2 expression or function

  • Transcriptomic profiling across cell types to correlate AK2 expression with specific cellular functions

  • Single-cell RNA sequencing to identify cell populations with distinctive AK2 expression patterns

• Proteomic approaches for AK2 interaction networks:

  • Affinity purification coupled with mass spectrometry using AK2 antibodies to identify interacting proteins

  • Proximity labeling methods (BioID, APEX) to map the AK2 protein neighborhood

  • Thermal proteome profiling to identify proteins affected by AK2 modulation

  • Cross-linking mass spectrometry to capture transient protein interactions

  • Phosphoproteomics to identify signaling pathways affected by AK2

• High-content imaging platforms:

  • Automated immunofluorescence with AK2 antibodies across cell lines or primary cells

  • Phenotypic profiling following AK2 modulation using automated image analysis

  • Live-cell tracking of mitochondrial parameters in AK2-modulated cells

  • Multi-parameter analysis correlating AK2 localization with cellular functions

• Metabolomic screening approaches:

  • Targeted metabolomics focusing on adenine nucleotides and energy metabolism

  • Untargeted metabolomic profiling to identify broader metabolic changes upon AK2 modulation

  • Stable isotope tracing to track metabolic flux in pathways connected to AK2 function

  • Integration of metabolomic data with transcriptomic and proteomic datasets

• Tissue and organism-level high-throughput analyses:

  • Tissue microarray analysis of AK2 expression across normal and pathological tissues

  • Model organism screens (zebrafish, Drosophila) with AK2 modifications

  • Patient-derived organoid screening for AK2-dependent phenotypes

  • High-throughput flow cytometry to analyze AK2's role in immune cell populations

• Drug and small molecule screening:

  • Compound library screening to identify modulators of AK2 expression or function

  • Targeted screens focusing on compounds affecting mitochondrial function or energy metabolism

  • Screening for molecules that can rescue phenotypes caused by AK2 deficiency

  • Identification of compounds that might synergize with AK2 modulation in disease contexts

By implementing these high-throughput methodological approaches, researchers can rapidly generate comprehensive datasets that illuminate AK2's diverse functions across different cellular contexts, potentially leading to new insights into its roles in normal physiology and disease states.