AK3L1 Antibody

What is AK3L1 and why is it significant in research?

AK3L1 (Adenylate Kinase 3-Like 1, also known as AK4) is a member of the adenylate kinase family of enzymes localized to the mitochondrial matrix. It plays a crucial role in regulating adenine and guanine nucleotide compositions within cells by catalyzing the reversible transfer of phosphate groups among nucleotides . The significance of AK3L1 in research stems from its tissue-specific expression pattern and developmental regulation. It is highly expressed in the kidney, moderately expressed in heart and liver, weakly expressed in brain, and barely detectable in placenta and lung . Studying AK3L1 provides insights into mitochondrial energy metabolism and nucleotide homeostasis, which are fundamental to cellular functions and may be disrupted in various pathological conditions.

What types of AK3L1 antibodies are currently available for research applications?

Researchers have access to several types of AK3L1/AK4 antibodies, each with specific characteristics:

• Mouse monoclonal antibodies:

  • Clone PSJB3-36AT: Derived from hybridization of mouse SP2/O myeloma cells with spleen cells from BALB/c mice immunized with recombinant human AK3L1

  • Clone OTI3E1: Produced against full-length human recombinant protein of AK4, showing reactivity with human, monkey, mouse, and rat samples

• Rabbit monoclonal antibodies:

  • Clone EPR7679: A recombinant monoclonal antibody available in various formats, including BSA and azide-free preparations

• Rabbit polyclonal antibodies:

  • Such as catalog #13206-1-AP, which targets AK3L1 and shows reactivity with human and mouse samples

These antibodies are designed for different experimental applications and provide researchers with options based on their specific needs, target species, and detection systems.

What are the common applications for AK3L1 antibodies in research settings?

AK3L1 antibodies can be employed in numerous research applications, with varying degrees of optimization required:

Each application requires specific optimization steps, including dilution testing, blocking conditions, and detection system compatibility assessment. Most antibodies perform robustly in Western blotting, while the other applications may require more extensive validation .

How should I design validation experiments when using AK3L1 antibodies for the first time?

Proper validation of AK3L1 antibodies is essential for ensuring reliable experimental results. A comprehensive validation approach should include:

• Positive and negative controls:

  • Positive controls: Use tissues/cells known to express AK3L1, such as kidney tissue, HepG2, Raji, or A431 cell lysates

  • Negative controls: Include tissues with minimal expression (placenta, lung) or use siRNA/shRNA knockdown models

• Cross-reactivity testing:

  • If working across species, verify cross-reactivity using samples from each target species

  • Compare reactivity patterns with published expression data

• Multi-technique validation:

  • Start with Western blot to confirm antibody specificity at the expected molecular weight (approximately 25-30 kDa)

  • Verify localization using IF/ICC (mitochondrial pattern expected)

  • Confirm tissue distribution patterns using IHC on appropriate tissue arrays

• Knockdown/knockout validation:

  • Use siRNA, CRISPR-Cas9, or shRNA approaches to reduce target expression

  • Compare antibody signal between wild-type and knockdown/knockout samples

This hierarchical validation approach ensures antibody specificity before proceeding to more complex or time-consuming experiments .

What are the optimal conditions for Western blot detection of AK3L1?

Western blot detection of AK3L1 requires careful optimization of several parameters:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • For mitochondrial proteins like AK3L1, consider mitochondrial enrichment protocols

  • Load 10-35 μg of total protein lysate per lane

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal resolution around 25-30 kDa

  • Transfer to PVDF or nitrocellulose membranes at 100V for 60-90 minutes

• Antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST

  • Primary antibody dilutions typically range from 1:500 to 1:2,000

  • For monoclonal antibodies, starting dilution of 1:1,000 is recommended

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

• Detection system:

  • Use HRP-conjugated secondary antibodies with ECL detection systems

  • Consider enhanced chemiluminescence for weak signals

• Expected results:

  • AK3L1 typically appears as a band at approximately 25-30 kDa

  • Multiple bands may indicate isoforms or post-translational modifications

Optimization of these parameters will help ensure consistent and reliable detection of AK3L1 in Western blot applications .

What are the critical considerations for immunohistochemical detection of AK3L1?

Immunohistochemical detection of AK3L1 requires attention to several critical factors:

• Tissue fixation and processing:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used

  • Fresh frozen sections may provide better epitope preservation but more challenging handling

• Antigen retrieval methods:

  • TE buffer (pH 9.0) is recommended for optimal retrieval

  • Alternative: citrate buffer (pH 6.0) may also be effective

  • Heat-induced epitope retrieval (pressure cooker or microwave) is typically necessary

• Antibody dilution and incubation:

  • Initial dilution range: 1:50-1:500

  • Incubate overnight at 4°C or 1-2 hours at room temperature

  • Use appropriate diluent with carrier protein to minimize background

• Detection system:

  • Polymer-based detection systems provide better sensitivity than ABC methods

  • DAB chromogen is standard for brightfield microscopy

  • Include counterstain (hematoxylin) for tissue context

• Controls:

  • Include kidney tissue as positive control (high expression)

  • Use isotype control antibodies for nonspecific binding assessment

  • Consider serial sections with primary antibody omission

These methodological considerations help ensure specific detection of AK3L1 in tissue sections while minimizing background and nonspecific staining .

How can AK3L1 antibodies be used to investigate mitochondrial function in disease models?

AK3L1 antibodies can serve as valuable tools for exploring mitochondrial dynamics and function in various disease models:

• Subcellular fractionation and localization studies:

  • Use AK3L1 antibodies as mitochondrial matrix markers

  • Combine with other mitochondrial compartment markers (outer membrane, intermembrane space, inner membrane) for comprehensive analysis

  • Investigate potential translocation under stress conditions using IF/ICC approaches

• Mitochondrial stress response:

  • Monitor AK3L1 expression changes during hypoxia, oxidative stress, or metabolic perturbations

  • Compare expression patterns with other mitochondrial proteins to establish regulatory relationships

  • Correlate AK3L1 levels with functional readouts of mitochondrial activity

• Tissue-specific comparisons:

  • Leverage the differential expression pattern of AK3L1 across tissues

  • Investigate kidney-specific functions given high expression levels

  • Compare expression in normal versus diseased tissues using IHC and WB

• Interaction studies:

  • Use co-immunoprecipitation with AK3L1 antibodies to identify binding partners

  • Investigate potential post-translational modifications affecting enzyme activity

  • Combine with proximity ligation assays for in situ protein interaction detection

These approaches enable researchers to investigate the role of AK3L1 in mitochondrial homeostasis and its potential dysregulation in disease states such as cancer, neurodegenerative disorders, and metabolic diseases .

What are the strategies for multiplexing AK3L1 detection with other mitochondrial markers?

Multiplexed detection of AK3L1 with other mitochondrial proteins provides comprehensive insights into mitochondrial biology. Several strategies can be employed:

• Multi-color immunofluorescence:

  • Select AK3L1 antibodies from different host species than other mitochondrial markers

  • Use fluorophore-conjugated secondary antibodies with non-overlapping emission spectra

  • Consider Tyramide Signal Amplification (TSA) for weak signals

  • Sequential staining protocols may be necessary to avoid cross-reactivity

• Chromogenic multiplexing in IHC:

  • Apply multiple primary antibodies sequentially

  • Use different chromogens (DAB, AEC, Fast Red) for distinct visualization

  • Careful optimization of antigen retrieval between cycles is critical

• Imaging mass cytometry or CODEX approaches:

  • Metal-conjugated antibodies allow detection of 40+ markers simultaneously

  • Requires specialized equipment but provides unprecedented multiplexing capacity

  • Consider unconjugated AK3L1 antibodies suitable for labeling

• Western blot multiplexing:

  • Use differently sized mitochondrial markers for simultaneous detection

  • Fluorescent secondary antibodies with different wavelengths

  • Sequential stripping and reprobing for antibodies from the same species

Each approach requires careful validation of antibody compatibility, potential interference, and signal specificity to ensure reliable multiplexed detection of mitochondrial markers including AK3L1 .

How can I troubleshoot specificity issues with AK3L1 antibodies in complex samples?

When encountering specificity issues with AK3L1 antibodies, systematic troubleshooting approaches should be implemented:

• Validation using genetic models:

  • CRISPR/Cas9 knockout or knockdown models provide definitive controls

  • Compare antibody reactivity patterns between wild-type and knockout samples

  • If knockout models aren't available, siRNA knockdown can serve as an alternative

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide/protein

  • Specific binding should be blocked, while nonspecific binding persists

  • Run parallel samples with and without peptide competition

• Cross-reactivity assessment:

  • Test antibody against recombinant proteins of similar family members

  • Consider potential cross-reactivity with AK3, which shares homology with AK3L1

  • Use tissues with differential expression of adenylate kinase family members

• Optimization strategies for reducing nonspecific binding:

  • Increase blocking agent concentration (5-10% BSA or milk)

  • Add detergents (0.1-0.3% Triton X-100) to reduce hydrophobic interactions

  • Optimize antibody concentration through careful titration

  • Consider alternative secondary antibodies if background persists

• Epitope accessibility issues:

  • For FFPE samples, test different antigen retrieval methods

  • For native proteins, ensure proper denaturation for Western blots

  • Consider alternative antibody clones targeting different epitopes

These systematic approaches can help resolve specificity issues and ensure reliable detection of AK3L1 in complex biological samples .

What control samples are essential when working with AK3L1 antibodies?

Rigorous control samples are fundamental to reliable AK3L1 antibody experiments:

• Positive tissue/cell controls:

  • Human kidney tissue (high expression)

  • HepG2, Raji, and A431 cell lines (confirmed expression)

  • Human fetal liver extracts (validated expression)

  • MCF-7 cells (validated for IP and IF applications)

• Negative/low expression controls:

  • Placenta and lung tissues (minimal expression)

  • Cell lines with low/no AK3L1 expression (verify through database searches)

• Technical controls:

  • Primary antibody omission control

  • Isotype-matched control antibodies

  • Secondary antibody-only controls

  • Non-specific IgG for IP experiments

• Genetic manipulation controls:

  • siRNA/shRNA knockdown samples

  • CRISPR/Cas9 knockout samples when available

  • Overexpression systems for positive control

• Species cross-reactivity controls:

  • When working with non-human samples, include human positive controls for comparison

  • Verify sequence homology between species for the antibody's target epitope

Including these diverse controls ensures experimental rigor and facilitates troubleshooting if unexpected results occur .

How should AK3L1 antibodies be stored and handled to maintain optimal performance?

Proper storage and handling of AK3L1 antibodies are essential for maintaining their performance over time:

• Long-term storage recommendations:

  • Store at -20°C for periods longer than one month

  • Shelf life typically extends to 12 months at -20°C when properly stored

  • Aliquot antibodies upon receipt to minimize freeze-thaw cycles

• Short-term storage:

  • For periods up to one month, 4°C storage is acceptable

  • Keep tightly sealed and protected from light

  • Return to -20°C for longer periods

• Critical handling practices:

  • Prevent freeze-thaw cycles which can degrade antibody performance

  • When removing from storage, thaw on ice or at 4°C, never at room temperature

  • Centrifuge briefly before opening to collect solution at the bottom of the tube

  • Use sterile technique when accessing antibody solutions

• Working solution preparation:

  • Dilute only the amount needed for immediate use

  • Use high-quality diluents (PBS with 0.1% BSA or carrier protein)

  • For PBS with 0.02% sodium azide and 50% glycerol formulations, ensure proper mixing

• Performance monitoring:

  • Include standard positive controls in each experiment

  • Monitor signal intensity over time

  • Document lot numbers and correlate with experimental outcomes

Adhering to these storage and handling practices will help maintain antibody performance and extend useful shelf life .

What are the key differences between monoclonal and polyclonal AK3L1 antibodies for research applications?

Understanding the differences between monoclonal and polyclonal AK3L1 antibodies is crucial for selecting the appropriate reagent:

For critical quantitative experiments requiring high reproducibility, monoclonal antibodies like PSJB3-36AT or EPR7679 are often preferred . For applications like IHC or IP where signal amplification is beneficial, polyclonal antibodies may provide advantages . The choice should be guided by the specific experimental requirements, target species, and application requirements.

How can I quantitatively analyze AK3L1 expression across different experimental conditions?

Quantitative analysis of AK3L1 expression requires rigorous methodological approaches:

• Western blot quantification:

  • Use appropriate loading controls (β-actin for whole cell lysates, VDAC or TOM20 for mitochondrial fractions)

  • Apply densitometric analysis with software like ImageJ or Image Lab

  • Normalize AK3L1 signal to loading control within linear detection range

  • Include standard curves using recombinant protein for absolute quantification

  • Present data as fold-change relative to control conditions

• qRT-PCR correlation studies:

  • Measure AK3L1 mRNA levels in parallel with protein detection

  • Design primers specific to AK3L1 (avoiding cross-amplification of AK3)

  • Normalize to stable reference genes appropriate for the experimental condition

  • Compare protein-mRNA correlations across conditions

• Immunofluorescence quantification:

  • Use consistent acquisition parameters (exposure time, gain)

  • Apply automated image analysis for unbiased quantification

  • Measure integrated density or mean fluorescence intensity

  • Normalize to mitochondrial mass using mitochondrial markers

  • Present data from multiple fields and biological replicates

• Statistical analysis requirements:

  • Perform experiments with at least three biological replicates

  • Apply appropriate statistical tests (t-test, ANOVA, non-parametric alternatives)

  • Report effect sizes along with p-values

  • Use multiple comparison corrections when appropriate

These approaches enable robust quantitative analysis of AK3L1 expression changes across experimental conditions, providing insights into regulatory mechanisms and functional significance .

What approaches can resolve contradictory results between different AK3L1 antibodies?

When different AK3L1 antibodies yield contradictory results, systematic investigation is necessary:

• Epitope mapping analysis:

  • Identify the specific epitopes recognized by each antibody

  • Determine if epitopes may be differentially accessible in various sample types

  • Consider potential post-translational modifications that might affect epitope recognition

  • Evaluate species-specific sequence variations at epitope regions

• Validation hierarchy implementation:

  • Establish a "gold standard" using genetic approaches (CRISPR knockout)

  • Compare antibody performance against this definitive control

  • Rank antibodies by specificity, sensitivity, and reproducibility

  • Consider antibody isotype and host species as factors in performance differences

• Application-specific optimization:

  • Recognize that an antibody performing well in WB may fail in IHC

  • Optimize each antibody individually for specific applications

  • Document application-specific dilutions and conditions for each antibody

• Combinatorial approaches:

  • Use multiple antibodies targeting different epitopes in parallel

  • Consider concordant results more reliable than single-antibody data

  • For critical findings, validate with orthogonal non-antibody methods

• Literature reconciliation:

  • Review published literature for similar discrepancies

  • Contact antibody manufacturers for technical support and known limitations

  • Consider contributing to public antibody validation repositories

This systematic approach helps resolve contradictions and establishes reliable protocols for studying AK3L1 expression and function .

How can AK3L1 antibodies be integrated into multi-omics research approaches?

Integration of AK3L1 antibody-based techniques with other omics approaches provides comprehensive insights:

• Proteomics integration:

  • Use AK3L1 antibodies for immunoprecipitation followed by mass spectrometry

  • Identify interaction partners and post-translational modifications

  • Compare antibody-based quantification with label-free or labeled proteomics data

  • Validate mass spectrometry findings with targeted antibody approaches

• Transcriptomics correlation:

  • Compare protein expression (antibody-based) with RNA-seq data

  • Investigate potential post-transcriptional regulation mechanisms

  • Identify concordant and discordant expression patterns across conditions

  • Use antibodies to validate key findings from transcriptomic studies

• Metabolomics connections:

  • Correlate AK3L1 expression with adenylate kinase activity measurements

  • Measure changes in substrate/product metabolites (AMP, ADP, ATP)

  • Investigate links between AK3L1 expression and broader metabolic alterations

  • Use antibodies to confirm causal relationships identified in metabolomic studies

• Spatial multi-omics:

  • Apply multiplex immunofluorescence with AK3L1 antibodies

  • Combine with in situ hybridization for spatial transcriptomics

  • Integrate with imaging mass spectrometry for spatial metabolomics

  • Develop computational methods to integrate multi-layer spatial data

• Functional genomics validation:

  • Use AK3L1 antibodies to validate CRISPR screen hits

  • Confirm protein-level changes in genetic perturbation studies

  • Apply ChIP-seq with transcription factor antibodies to study AK3L1 regulation

  • Develop reporter assays to validate regulatory mechanisms

These integrated approaches leverage the specificity of antibody-based detection while gaining broader systems-level insights into AK3L1 biology and function .

What emerging technologies might enhance AK3L1 antibody applications in research?

Several emerging technologies show promise for enhancing AK3L1 antibody applications:

• Single-cell protein analysis:

  • Mass cytometry adaptations for intracellular AK3L1 detection

  • Microfluidic approaches for single-cell Western blotting

  • Integration with single-cell transcriptomics for multi-omic profiling

  • Nanobody-based detection systems for improved penetration

• Super-resolution microscopy:

  • STORM/PALM techniques for nanoscale localization within mitochondria

  • Expanded microscopy for physical sample expansion and improved resolution

  • Correlative light-electron microscopy for ultrastructural context

  • Live-cell super-resolution imaging with minimally disruptive antibody fragments

• Antibody engineering approaches:

  • Recombinant antibody fragments with superior tissue penetration

  • Site-specific conjugation for improved fluorophore performance

  • Bifunctional antibodies for proximity detection applications

  • CRISPR-generated knock-in epitope tags for endogenous protein detection

• Proximity-based assay adaptations:

  • Antibody-based proximity ligation assays for protein interactions

  • Split-enzyme complementation systems for dynamic interaction studies

  • APEX2 peroxidase fusions for proximity biotinylation proteomics

  • HaloTag and SNAP-tag systems for orthogonal labeling strategies

These emerging technologies will likely expand the utility of AK3L1 antibodies beyond current applications, enabling more sensitive detection, better spatial resolution, and more comprehensive understanding of AK3L1 biology .

What are the most promising research directions involving AK3L1 antibodies in disease studies?

AK3L1 antibodies are poised to contribute to several promising disease-related research directions:

• Cancer metabolism studies:

  • Investigation of AK3L1 expression changes in tumors versus normal tissues

  • Correlation with hypoxia markers and metabolic reprogramming

  • Potential prognostic value in specific cancer types

  • Target validation for metabolic intervention strategies

• Neurodegenerative disease research:

  • Examination of mitochondrial dysfunction in Alzheimer's and Parkinson's

  • Analysis of AK3L1 expression in affected brain regions

  • Correlation with markers of oxidative stress and energy failure

  • Investigation as a potential biomarker for disease progression

• Kidney disease mechanisms:

  • Leveraging high kidney expression for specific pathophysiological studies

  • Analysis in models of acute kidney injury and chronic kidney disease

  • Investigation of tubular epithelial cell energetics and AK3L1 function

  • Potential therapeutic target in mitochondrial dysfunction

• Cardiovascular disease pathways:

  • Study of AK3L1 in cardiomyocyte adaptation to stress

  • Role in heart failure progression and metabolic remodeling

  • Potential involvement in ischemia-reperfusion injury mechanisms

  • Investigation in cardiac hypertrophy models

• Metabolic disease connections:

  • Analysis in models of diabetes and metabolic syndrome

  • Investigation of AK3L1 regulation by insulin and metabolic sensors

  • Potential contribution to mitochondrial adaptations in obesity

  • Role in tissue-specific metabolic reprogramming

These research directions leverage AK3L1 antibodies as tools to uncover novel disease mechanisms, potential biomarkers, and therapeutic targets across multiple pathological conditions .

How might standardization efforts improve reproducibility in AK3L1 antibody-based research?

Standardization initiatives could substantially improve research reproducibility with AK3L1 antibodies:

• Antibody validation standards:

  • Implementation of knockout-validated antibody criteria

  • Multi-laboratory validation of commercially available antibodies

  • Development of application-specific validation protocols

  • Creation of shared positive and negative control materials

• Reporting requirements enhancement:

  • Detailed documentation of antibody catalog numbers and lot information

  • Standardized reporting of dilutions, incubation conditions, and detection methods

  • Publication of validation data alongside experimental results

  • Inclusion of all control experiments in supplementary materials

• Reference standard development:

  • Creation of recombinant AK3L1 reference materials

  • Development of standardized lysates with defined AK3L1 quantities

  • Production of synthetic peptide standards for epitope-specific validation

  • Establishment of digital reference images for IHC/IF interpretation

• Protocol harmonization:

  • Development of consensus protocols for common applications

  • Interlaboratory testing of protocol robustness

  • Creation of detailed troubleshooting guidelines

  • Establishment of quality control metrics for each application

• Data repository contributions:

  • Submission of antibody validation data to public repositories

  • Sharing of raw unprocessed images in publications

  • Development of antibody performance tracking systems

  • Integration with broader reproducibility initiatives in biomedical research

These standardization efforts would enhance confidence in AK3L1 antibody-based research findings, facilitate cross-study comparisons, and accelerate scientific progress in understanding this important mitochondrial enzyme .