AK1 Antibody

What is AK1 and why is it a significant research target?

Adenylate Kinase 1 (AK1) is an enzyme that catalyzes the reversible transfer of the terminal phosphate group between ATP and AMP. It plays a crucial role in cellular energy homeostasis and adenine nucleotide metabolism . AK1 is particularly significant as a research target because it serves as an important marker in various cellular processes and pathological conditions, including cancer. Recent studies have shown that AK1 expression levels may have prognostic value in acute myeloid leukemia (AML) patients undergoing chemotherapy . When investigating AK1, researchers must consider its tissue distribution, with high expression observed in skeletal muscle, heart, and brain tissues .

What types of AK1 antibodies are available for research applications?

Several types of AK1 antibodies are available for research purposes, including:

• Polyclonal antibodies: Typically rabbit-derived affinity-purified anti-AK1 antibodies that recognize multiple epitopes of the AK1 protein

• Monoclonal antibodies: Mouse-derived anti-AK1 IgG1 Kappa antibodies that target specific epitopes with high specificity

• Matched antibody pairs: Sets containing both capture (typically rabbit polyclonal) and detection (typically mouse monoclonal) antibodies designed specifically for ELISA applications

The selection of antibody type depends on the specific application, with polyclonal antibodies offering broader epitope recognition but potentially higher background, while monoclonal antibodies provide higher specificity but may be less robust to sample preparation variations.

What are the main applications for AK1 antibodies in research?

AK1 antibodies have been validated for multiple research applications including:

Each application requires specific optimization steps to ensure reliable results, including antibody titration, blocking optimization, and appropriate controls .

How should AK1 antibodies be stored and handled to maintain reactivity?

Proper storage and handling of AK1 antibodies are critical for maintaining their reactivity and specificity. Recommended practices include:

• Store antibodies at -20°C or lower to preserve activity over extended periods

• Aliquot antibodies into smaller volumes upon receipt to avoid repeated freeze-thaw cycles, which can degrade antibody performance

• Return reagents to -20°C storage immediately after use

• When working with antibody pairs, store capture and detection antibodies separately to prevent cross-contamination

• Follow manufacturer-specific recommendations for reconstitution of lyophilized antibodies

• Monitor expiration dates and validate antibody performance periodically, especially for critical experiments

Proper handling significantly extends antibody shelf-life and ensures consistent experimental results across multiple studies.

How can researchers validate the specificity of AK1 antibodies in their experimental systems?

Antibody validation is critical for ensuring experimental reproducibility. For AK1 antibodies, comprehensive validation should include:

• Genetic strategies: Testing antibody reactivity in AK1 knockout/knockdown models versus wild-type samples to confirm specificity. This can be accomplished through CRISPR-Cas9 mediated gene editing or siRNA approaches .

• Orthogonal strategies: Comparing antibody-based detection with non-antibody-based methods such as mass spectrometry or RNA-seq to confirm target expression patterns .

• Independent antibody verification: Using multiple antibodies targeting different epitopes of AK1 to confirm consistent detection patterns .

• Expression validation: Testing antibody in tissues/cells known to express high levels of AK1 (e.g., skeletal muscle, heart) versus those with minimal expression .

• Recombinant protein controls: Using purified recombinant AK1 protein as a positive control and conducting competition assays with the target antigen .

A comprehensive validation approach combines at least two of these methods to ensure confidence in antibody specificity before proceeding with experimental applications.

What are the key considerations when using AK1 antibodies for investigating protein-protein interactions?

When investigating AK1 protein-protein interactions, researchers should consider:

• Native conditions preservation: Choose immunoprecipitation protocols that maintain native protein conformations to preserve physiological interactions. Mild detergents like NP-40 or Triton X-100 at 0.1-0.5% are often suitable .

• Cross-linking considerations: For transient interactions, consider using reversible cross-linkers like DSP (dithiobis[succinimidylpropionate]) to stabilize complexes before immunoprecipitation.

• Antibody orientation: For co-immunoprecipitation, determine whether the AK1 antibody should be used as the capture antibody (to pull down AK1 and associated proteins) or for detection of AK1 in complexes pulled down by antibodies against potential interaction partners .

• Validation controls: Include appropriate controls such as IgG control immunoprecipitations and lysates from cells with AK1 knockdown/knockout .

• Quantification methods: Consider quantitative approaches like SWATH-MS (Sequential Window Acquisition of All Theoretical Mass Spectra) to identify and quantify interaction partners.

• Physiological relevance: Validate interactions identified in vitro through cellular co-localization studies using immunofluorescence with the AK1 antibody and antibodies against potential interaction partners.

How can AK1 antibodies be utilized to investigate its role in disease mechanisms?

AK1 has been implicated in various disease mechanisms, particularly in cancer and metabolic disorders. Researchers can utilize AK1 antibodies to investigate these associations through:

• Prognostic marker analysis: As demonstrated in acute myeloid leukemia studies, AK1 expression levels may correlate with patient outcomes. Immunohistochemistry with validated AK1 antibodies can be used on patient tissue microarrays to establish correlations with clinical parameters .

• Signaling pathway investigations: AK1 functions in energy homeostasis pathways that may be dysregulated in disease. Phospho-specific antibodies against proteins in related pathways (e.g., AMPK, mTOR) can be used alongside AK1 antibodies to map altered signaling networks.

• Therapeutic response monitoring: Changes in AK1 expression or localization following treatment can be monitored using antibody-based approaches like western blotting, IHC, or immunofluorescence to assess therapy effectiveness.

• Subcellular localization studies: Alterations in AK1 subcellular distribution in disease states can be investigated using fractionation techniques followed by western blotting or through high-resolution microscopy with fluorescently-labeled AK1 antibodies.

• Biomarker development: For diseases where AK1 shows altered expression, antibody-based assays such as tissue microarray analysis or serum ELISA could be developed for diagnostic applications.

The recent finding that high AK1 expression predicts inferior prognosis in AML patients undergoing chemotherapy highlights the potential value of AK1 as a prognostic biomarker that could influence treatment decisions .

What are the optimized protocols for using AK1 antibodies in Western blotting?

For optimal Western blot results with AK1 antibodies, researchers should follow these methodological guidelines:

• Sample preparation:

  • For tissue samples: Homogenize in RIPA buffer supplemented with protease inhibitors

  • For cell lines: Lyse in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and protease inhibitors

  • Load 20-30 μg of total protein per lane based on validated protocols

• Electrophoresis and transfer:

  • Use reducing conditions (include β-mercaptoethanol or DTT in sample buffer)

  • Run on 12-15% SDS-PAGE gels (AK1 has a predicted band size of 22 kDa)

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

• Antibody incubation:

  • Block membrane in 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with primary AK1 antibody at 1:5000-1:10000 dilution overnight at 4°C

  • Wash 3-5 times with TBST

  • Incubate with HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

• Detection:

  • Develop using enhanced chemiluminescence (ECL) technique

  • Expected band: 22 kDa (confirm with positive control such as skeletal muscle or heart tissue lysate)

• Validation controls:

  • Positive control: Mouse skeletal muscle or heart tissue lysate

  • Negative control: Tissues with minimal AK1 expression

  • Specificity control: AK1 knockdown/knockout cell lysates

How can researchers troubleshoot common issues with AK1 antibody-based ELISA assays?

ELISA assays using matched AK1 antibody pairs may encounter several common issues. Here are troubleshooting approaches for each:

Researchers should determine the optimal working dilution for each new lot of antibody through titration experiments, as recommended by manufacturers .

What considerations should be made when designing immunohistochemistry experiments with AK1 antibodies?

Successful immunohistochemistry (IHC) with AK1 antibodies requires attention to several critical parameters:

• Tissue fixation and processing:

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

  • Fixation time affects epitope accessibility; standardize fixation protocols

  • Consider performing antigen retrieval optimization experiments (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

• Antibody selection and validation:

  • Verify the antibody has been validated for IHC applications

  • Polyclonal antibodies often perform well in IHC due to recognition of multiple epitopes

  • Dilution ranges typically start at 1:100-1:250 for AK1 antibodies

• Positive and negative controls:

  • Positive tissue controls: Human skeletal muscle and heart tissues (high AK1 expression)

  • Negative controls: Include sections with primary antibody omitted

  • Consider using tissue with known differential expression patterns

• Detection systems:

  • For low abundance targets, consider signal amplification methods (e.g., tyramide signal amplification)

  • Chromogenic detection (DAB) versus fluorescent detection depends on research needs

  • Multiplex IHC may require specialized fluorescent secondary antibodies

• Quantification methods:

  • Define clear scoring systems for AK1 expression (e.g., H-score, percentage positive cells)

  • Consider digital pathology approaches for unbiased quantification

  • For prognostic studies, establish cutoff values based on clinical outcomes

Published protocols have successfully used AK1 antibodies at 1:100 dilution for paraffin-embedded human skeletal muscle and thyroid tissue , but optimization is recommended for each new tissue type.

How are AK1 antibodies being applied in cancer research and biomarker development?

AK1 antibodies are increasingly utilized in cancer research across multiple applications:

• Prognostic biomarker development: Recent studies demonstrated that high AK1 expression predicts inferior prognosis in acute myeloid leukemia patients undergoing chemotherapy, suggesting AK1 as a potential biomarker for treatment stratification . AK1 antibodies enable researchers to:

  • Quantify AK1 expression in patient samples using IHC or tissue microarrays

  • Correlate expression levels with clinical outcomes and treatment responses

  • Develop standardized scoring systems for potential clinical implementation

• Metabolic reprogramming investigations: Cancer cells often exhibit altered energy metabolism (Warburg effect). AK1 antibodies help researchers:

  • Map changes in energy metabolism enzymes across cancer types

  • Investigate AK1's relationship with other metabolic enzymes in tumor microenvironments

  • Study how metabolic adaptations contribute to treatment resistance

• Therapeutic target validation: As metabolism-targeting therapies gain interest, AK1 antibodies facilitate:

  • High-throughput screening of compounds that modulate AK1 expression or activity

  • Mechanism-of-action studies for metabolic pathway inhibitors

  • Patient selection strategies for metabolism-targeted therapies

• Liquid biopsy development: Emerging research is exploring whether AK1 could serve as a circulating biomarker, with antibody-based assays enabling:

  • Detection of AK1 in patient serum or plasma

  • Monitoring of treatment response through changes in circulating AK1 levels

  • Development of multiplexed assays combining AK1 with other biomarkers

The correlation between AK1 expression and clinical outcomes in AML represents an important direction for future research, potentially expanding to other cancer types .

What are the latest innovations in antibody validation that researchers should apply to AK1 antibodies?

Antibody validation technologies continue to evolve, with several cutting-edge approaches that should be considered for AK1 antibodies:

• Multiomics validation approaches:

  • Integration of antibody-based detection with transcriptomics and proteomics

  • Correlation of protein levels detected by antibodies with mRNA expression data

  • Validation through mass spectrometry-based proteomics to confirm specificity

• Advanced genetic validation:

  • CRISPR-Cas9 engineered cell lines with tagged endogenous AK1

  • Inducible knockout/knockin systems for temporal control of validation

  • Isogenic cell line panels differing only in AK1 expression levels

• High-throughput validation platforms:

  • Protein arrays for cross-reactivity testing across thousands of proteins

  • Tissue microarrays for simultaneous validation across multiple tissue types

  • Automated imaging and quantification systems for standardized evaluation

• Enhanced reproducibility frameworks:

  • Standardized reporting of validation data using MDAR (Materials, Design, Analysis and Reporting) guidelines

  • Repository submissions of validation data to community resources

  • Independent validation through multicenter testing

• AI-assisted validation tools:

  • Machine learning algorithms for predicting antibody specificity

  • Automated image analysis to detect non-specific binding patterns

  • Computational approaches to identify optimal epitopes for antibody generation

These advanced validation approaches move beyond traditional methods to provide more comprehensive evidence of antibody specificity and reliability .

How can AK1 antibodies be integrated into multiplexed detection systems for comprehensive pathway analysis?

Modern research increasingly requires simultaneous detection of multiple targets for pathway analysis. AK1 antibodies can be integrated into multiplexed systems through:

• Multiplexed immunofluorescence techniques:

  • Sequential multiplexing using tyramide signal amplification (TSA)

  • Spectral unmixing to resolve overlapping fluorophores

  • Panel design considerations to include AK1 alongside energy metabolism markers (e.g., AMPK, mTOR, HK2)

• Mass cytometry approaches:

  • Metal-conjugated AK1 antibodies for CyTOF (Cytometry by Time of Flight) analysis

  • Integration into panels for simultaneous detection of 30+ proteins

  • Single-cell resolution of AK1 expression in heterogeneous samples

• Spatial transcriptomics integration:

  • Combining antibody-based protein detection with in situ RNA analysis

  • Correlating AK1 protein levels with transcript expression at single-cell resolution

  • Mapping spatial relationships between AK1 and interacting partners

• Automated multiplexed IHC systems:

  • Optimization of AK1 antibodies for platforms like Vectra/Polaris

  • Development of standardized multiplexed panels including AK1

  • Quantitative spatial analysis of AK1 in the context of tumor microenvironment

• Bead-based multiplex assays:

  • Adaptation of AK1 antibodies for suspension array technologies

  • Development of multiplex ELISAs including AK1 and related proteins

  • High-throughput screening applications for drug discovery

These multiplexed approaches enable researchers to position AK1 within its broader signaling context, providing more comprehensive insights into its biological roles and disease associations.