AK2 Antibody

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

AK2 (adenylate kinase 2) is a critical enzyme that catalyzes the reversible transfer of terminal phosphate groups between ATP and AMP, playing an essential role in cellular energy homeostasis and adenine nucleotide metabolism. Its primary localization in the mitochondrial intermembrane space makes it a key regulator of ATP levels and cellular metabolism. AK2 is particularly important in hematopoiesis and maintaining mitochondrial function under various cellular conditions. Dysregulation of AK2 activity has been linked to metabolic disorders, neurodegenerative diseases, and cancer, making it an important research target for understanding these pathological conditions .

What types of AK2 antibodies are available for research?

Several types of AK2 antibodies are available for research purposes, each with specific characteristics:

These antibodies differ in specificity, sensitivity, and application compatibility, allowing researchers to select the most appropriate tool for their specific experimental needs .

How do I select the appropriate AK2 antibody for my experiment?

When selecting an AK2 antibody, consider the following methodological approach:

• Determine your experimental application (WB, IHC, IF, IP, ELISA, Flow Cytometry)

• Identify your sample species (human, mouse, rat) and ensure cross-reactivity

• Consider antibody format (unconjugated vs. conjugated with HRP, PE, FITC, or Alexa Fluor)

• Evaluate clonality requirements (polyclonal antibodies offer broader epitope recognition, while monoclonal antibodies provide higher specificity and reproducibility)

• Review validation data (published applications, knockout validation)

• Consider storage conditions and stability requirements

For example, if performing Western blot analysis on human samples, both polyclonal (11014-1-AP) and monoclonal (F-2) antibodies would be suitable, but the monoclonal might provide more consistent results across experiments .

What are the recommended dilutions for AK2 antibodies in common applications?

The optimal dilution of AK2 antibodies varies by application and specific antibody. Based on validated protocols, the following dilutions are recommended:

It is strongly recommended to titrate each antibody in your specific experimental system to obtain optimal signal-to-noise ratios. Sample-dependent optimization may be necessary to account for variations in AK2 expression levels across different cell types and tissues .

How should I optimize Western blot protocols for AK2 detection?

For optimal Western blot detection of AK2 (26 kDa), follow these methodological steps:

• Sample preparation: Use RIPA buffer for protein extraction from cells or tissues

• SDS-PAGE: Use 12% polyacrylamide gels for optimal resolution of the 26 kDa AK2 protein

• Transfer: Standard PVDF membranes are suitable for AK2 transfer

• Blocking: Block with PBST containing 5% non-fat milk at 25°C for 60 minutes

• Primary antibody: Incubate with AK2 antibody at recommended dilution (1:1000-1:4000 for 11014-1-AP) at 4°C overnight

• Secondary antibody: Use appropriate HRP-conjugated secondary antibody (anti-rabbit or anti-mouse depending on primary antibody host)

• Detection: Develop with ECL Plus or similar chemiluminescence reagent

• Controls: Include β-actin as a loading control and consider using AK2 knockout samples as negative controls when available

For detecting subtle changes in AK2 expression, the recombinant monoclonal antibody EPR11387(B) may provide higher sensitivity and specificity compared to polyclonal alternatives .

What are the key considerations for immunohistochemical detection of AK2?

When performing immunohistochemistry for AK2 detection, consider these critical methodological points:

• Antigen retrieval: Use TE buffer at pH 9.0 for optimal epitope exposure (alternatively, citrate buffer at pH 6.0 can be used)

• Antibody selection: For IHC applications, both 11014-1-AP (polyclonal) and F-2 (monoclonal) antibodies have been validated

• Dilution: Start with 1:50-1:500 dilution and optimize based on signal strength

• Tissue specificity: AK2 has been successfully detected in human pancreatic cancer tissue

• Controls: Include positive control tissues with known AK2 expression (liver, kidney) and negative controls (primary antibody omission)

• Counterstaining: Use appropriate nuclear counterstains to facilitate cellular localization analysis

• Visualization: Look for mitochondrial intermembrane space localization pattern, which is characteristic of AK2

Remember that tissue fixation conditions can significantly impact epitope accessibility, so optimization may be required for different fixation protocols .

How can I study AK2 translocation during apoptosis?

AK2 translocates from the mitochondrial intermembrane space to the cytosol during early apoptotic events, alongside cytochrome c. To effectively study this translocation:

• Experimental design:

  • Induce apoptosis using appropriate stimuli (e.g., staurosporine, TNF-α)

  • Collect samples at multiple time points (early, mid, and late apoptosis)

  • Prepare subcellular fractions (cytosolic, mitochondrial)

• Detection methods:

  • Immunofluorescence: Use AK2 Antibody F-2 (1:50-1:500) to visualize real-time translocation

  • Co-staining: Combine with cytochrome c antibodies to confirm apoptotic events

  • Western blot: Analyze subcellular fractions for AK2 redistribution

  • Flow cytometry: For quantitative assessment of translocation in cell populations

• Controls and validation:

  • Use apoptosis inhibitors to confirm specificity of translocation

  • Include mitochondrial markers (e.g., COX IV) and cytosolic markers (e.g., GAPDH) to verify fractionation quality

  • Consider AK2 knockdown or knockout controls to validate antibody specificity

This approach allows for comprehensive analysis of AK2's dynamic role in apoptotic signaling pathways and mitochondrial function during cellular stress .

What is the significance of AK2 as a biomarker, and how can it be validated?

AK2 has emerging significance as a potential biomarker in various diseases. To validate AK2 as a biomarker:

• Quantitative assessment methods:

  • Proteomic analysis: Use mass spectrometry for unbiased quantification

  • Western blot: For targeted validation of proteomic findings

  • ELISA: For high-throughput quantification in clinical samples

  • Immunohistochemistry: For tissue-specific expression analysis

• Validation workflow:

  • Discovery phase: Identify AK2 expression changes in disease vs. control samples

  • Verification phase: Confirm findings in independent sample sets

  • Statistical analysis: Apply appropriate tests (e.g., Mann-Whitney) for significance assessment

  • Correlation analysis: Link AK2 expression to clinical parameters and outcomes

• Control considerations:

  • Include multiple loading controls for normalization

  • Account for cell/tissue heterogeneity in samples

  • Consider confounding factors such as medication effects

For example, in a proteomic study using T cells, researchers identified AK2 as a potential biomarker and confirmed findings using immunoblotting with Santa Cruz AK2 antibody (sc-28,786) at 1:100 dilution. The analysis included proper controls and statistical validation using the Mann-Whitney test with p<0.05 considered significant .

How can I investigate AK2's role in metabolic disorders using antibody-based approaches?

To investigate AK2's role in metabolic disorders:

• Expression profiling strategy:

  • Compare AK2 levels across different metabolic states using Western blot (1:1000-1:4000 dilution)

  • Analyze tissue-specific expression patterns using IHC (1:50-1:500 dilution)

  • Assess subcellular localization changes using IF (1:50-1:500 dilution)

• Functional analysis approaches:

  • Co-immunoprecipitation: Use 0.5-4.0 μg of AK2 antibody for 1.0-3.0 mg of total protein to identify AK2 interaction partners

  • Proximity ligation assay: To detect in situ protein-protein interactions

  • Enzymatic activity correlation: Link AK2 expression levels with adenylate kinase activity measurements

• Disease model applications:

  • Apply these methods to relevant disease models (e.g., diabetes, obesity, mitochondrial disorders)

  • Analyze AK2 expression in patient samples with metabolic dysfunction

  • Correlate findings with clinical parameters and disease progression

This comprehensive approach can reveal how AK2 dysfunction contributes to metabolic disorders through alterations in energy metabolism and mitochondrial function .

How do I troubleshoot weak or non-specific AK2 antibody signals in Western blots?

When encountering weak or non-specific signals in AK2 Western blots, implement this systematic troubleshooting approach:

• For weak signals:

  • Increase primary antibody concentration (start with 2-fold increase)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Increase protein loading (up to 50-75 μg per lane)

  • Use enhanced chemiluminescence detection systems

  • Consider sample preparation modifications to enhance AK2 extraction

• For non-specific bands:

  • Increase blocking stringency (5% BSA instead of milk)

  • Add 0.1-0.5% Tween-20 to washing buffer

  • Reduce primary antibody concentration

  • Increase washing time and frequency

  • Try a different AK2 antibody (monoclonal F-2 for higher specificity)

• Validation steps:

  • Run AK2 knockout/knockdown control samples if available

  • Check expected molecular weight (26 kDa for AK2)

  • Consider tissue/cell type-specific expression patterns

For example, when using 11014-1-AP antibody, researchers have successfully detected AK2 in HL-60 cells, HeLa cells, HepG2 cells, and mouse/rat kidney and liver tissues, suggesting these as potential positive controls .

What controls should be included when studying AK2 in different experimental systems?

To ensure experimental rigor when studying AK2, incorporate these essential controls:

• Positive controls:

  • Cell lines with confirmed AK2 expression: HL-60, HeLa, HepG2

  • Tissue samples: Mouse/rat liver and kidney tissues

  • Recombinant AK2 protein (for antibody validation)

• Negative controls:

  • AK2 knockout cell lines (e.g., Human AK2 knockout HEK-293T cell line ab266539)

  • Primary antibody omission controls

  • Isotype controls for immunostaining

• Technical controls:

  • Loading controls: β-actin, GAPDH

  • Subcellular fraction markers: COX IV (mitochondria), GAPDH (cytosol)

  • Signal specificity: Peptide competition assays

• Application-specific controls:

  • For IHC: Adjacent section controls with normal tissue

  • For IP: IgG control pulldowns

  • For IF: Secondary antibody-only controls

Implementing these controls ensures reliable interpretation of results and helps distinguish true AK2 signals from experimental artifacts .

How do I interpret conflicting data regarding AK2 expression or localization?

When faced with conflicting data regarding AK2 expression or localization:

• Methodological reconciliation:

  • Compare antibody clones used (different epitopes may yield different results)

  • Evaluate fixation and preparation methods (may affect epitope accessibility)

  • Consider detection techniques (sensitivity differences between methods)

  • Examine subcellular fractionation procedures for potential cross-contamination

• Biological considerations:

  • Cell type-specific expression patterns (AK2 levels vary across tissues)

  • Dynamic localization during cellular processes (AK2 translocates during apoptosis)

  • Post-translational modifications affecting antibody recognition

  • Splice variants or isoforms with different cellular distribution

• Validation approaches:

  • Use multiple, independent antibodies targeting different epitopes

  • Employ complementary techniques (WB, IF, IHC, mass spectrometry)

  • Conduct genetic validation (siRNA knockdown, CRISPR knockout)

  • Perform functional assays to correlate protein detection with activity

For example, apparent discrepancies in AK2 localization might be explained by its dynamic translocation from mitochondria to cytosol during apoptosis, a biological process rather than a technical artifact .

How can AK2 antibodies be used to study hematopoiesis and related disorders?

AK2 plays a key role in hematopoiesis, making it an important target for studying blood cell development and related disorders:

• Experimental approaches:

  • Expression profiling: Use Western blot (1:1000-1:4000 dilution) to quantify AK2 levels in different hematopoietic cell populations

  • Immunophenotyping: Combine AK2 staining with hematopoietic lineage markers in flow cytometry

  • Developmental analysis: Track AK2 expression during differentiation of hematopoietic stem cells

• Disease model applications:

  • Reticular dysgenesis: Analyze AK2 expression in patient samples

  • Bone marrow failure syndromes: Compare AK2 levels in normal vs. pathological samples

  • Leukemia models: Investigate AK2 alterations in malignant transformation

• Mechanistic studies:

  • Co-localization with mitochondrial markers in hematopoietic cells

  • Analysis of AK2's role in energy metabolism during hematopoietic differentiation

  • Investigation of AK2-dependent signaling pathways in blood cell development

This research has significant implications for understanding congenital neutropenia, immunodeficiency, and other hematological disorders linked to AK2 dysfunction .

What is the relationship between AK2 and cancer, and how can antibodies help investigate this connection?

The relationship between AK2 and cancer can be investigated using antibody-based approaches:

• Expression analysis strategies:

  • Tissue microarray screening: Use IHC (1:50-1:500 dilution) to profile AK2 expression across various cancer types

  • Cancer cell line panels: Quantify AK2 by Western blot (1:1000-1:4000 dilution) in cancer vs. normal cell lines

  • Patient sample analysis: Compare AK2 levels in tumor vs. adjacent normal tissue

• Functional investigation approaches:

  • Proliferation correlation: Link AK2 expression to cancer cell growth rates

  • Metabolic phenotyping: Assess relationship between AK2 levels and metabolic reprogramming in cancer

  • Therapy response: Monitor AK2 expression changes following anti-cancer treatments

• Mechanistic studies:

  • Interaction partners: Identify cancer-specific AK2 protein complexes via co-immunoprecipitation

  • Subcellular distribution: Assess alterations in AK2 localization in cancer cells

  • Post-translational modifications: Investigate cancer-associated modifications of AK2

For example, researchers have successfully used AK2 antibodies to detect the protein in human pancreatic cancer tissue, demonstrating the utility of these tools in cancer research .

How can advanced imaging techniques be combined with AK2 antibodies for mitochondrial research?

Advanced imaging techniques can be powerfully combined with AK2 antibodies to study mitochondrial biology:

• Super-resolution microscopy approaches:

  • STED microscopy: Achieve 20-30 nm resolution to precisely localize AK2 within mitochondrial compartments

  • PALM/STORM: For single-molecule localization of AK2 in the mitochondrial intermembrane space

  • SIM: For 3D visualization of AK2 distribution in mitochondrial networks

• Live-cell imaging strategies:

  • Fluorescently conjugated AK2 antibodies (FITC, PE, Alexa Fluor) for dynamic studies

  • Proximity ligation assays to visualize AK2 interactions in situ

  • FRET-based approaches to study conformational changes and protein-protein interactions

• Correlative microscopy methods:

  • CLEM (Correlative Light and Electron Microscopy): Combine fluorescence imaging of AK2 with ultrastructural analysis

  • Cryo-electron tomography with immunogold labeling: For high-resolution 3D localization

• Multiplexed imaging:

  • Co-staining of AK2 with other mitochondrial proteins to study spatial relationships

  • Sequential imaging of multiple targets to build comprehensive mitochondrial protein maps

These approaches can reveal novel insights into AK2's precise localization and dynamic behavior during various cellular processes, particularly during stress conditions and apoptosis .