AK1 Antibody

What is AK1 and what are its primary functions in cellular metabolism?

AK1 is a 22kDa phosphotransferase that catalyzes the reversible transfer of terminal phosphate groups between ATP and AMP. Beyond this primary function, AK1:

• Displays broad nucleoside diphosphate kinase activity

• Plays an important role in cellular energy homeostasis and adenine nucleotide metabolism

• Can catalyze at very low rates the synthesis of thiamine triphosphate (ThTP) from thiamine diphosphate (ThDP) and ADP

• Shuttles ATP to energy-consuming sites within cells

AK1 is predominantly localized in the cytoplasm and is highly expressed in tissues with elevated energy demands, such as skeletal muscle and cardiac tissue. The protein's critical role in energy transfer pathways makes it an important subject for metabolic research.

What types of AK1 antibodies are currently available for research applications?

Several types of antibodies targeting AK1 are available to researchers:

Most commercially available antibodies are derived from immunogens containing sequences corresponding to specific amino acid regions of human AK1. For instance, the polyclonal antibody CAB1218 is generated against a recombinant fusion protein containing amino acids 1-194 of human AK1 (NP_000467.1) . Researchers should select antibodies based on their specific experimental needs and target species.

How do I validate the specificity of an AK1 antibody for my research?

Proper validation of AK1 antibodies requires a multi-faceted approach:

• Knockout/knockdown controls: Use AK1 knockout cell lines to confirm loss of signal specificity. For example, the anti-AK1 antibody [EP7083] has been validated using human AK1 knockout HeLa cell lines, demonstrating specific reactivity in wild-type cells and signal loss in knockout samples .

• Molecular weight confirmation: Verify detection at the expected molecular weight (22kDa for AK1).

• Loading controls: Run parallel detection of housekeeping proteins like GAPDH to ensure equal loading across samples.

• Cross-reactivity assessment: Test the antibody against other adenylate kinase isoforms to confirm specificity.

• Multiple detection methods: Confirm results across different techniques (e.g., WB, IF, IHC) when possible.

Methodologically, prepare wild-type and AK1 knockout samples using standard SDS-PAGE protocols, and incubate with your antibody at the manufacturer's recommended dilution (typically 1:500-1:2000 for Western blotting).

What experimental considerations should be made when using AK1 antibodies for localization studies in specialized cell types?

When investigating AK1 localization in specialized cells, researchers should consider:

• Cell-type specific localization patterns:

  • In most cells: AK1 is primarily cytoplasmic

  • In spermatozoa: AK1 localizes to whole flagella (plasmalemma, mitochondria, outer dense fibers)

  • In cardiac cells: AK1 may associate with both cytosolic and membrane-bound compartments

• Fixation optimization: Different fixation protocols affect epitope accessibility. For membrane-associated AK1, paraformaldehyde (4%) is often preferred over methanol fixation.

• Permeabilization conditions: Adjust based on subcellular target location (mild for membrane proteins, stronger for cytoskeletal associations).

• Co-localization studies: Use established organelle markers alongside AK1 antibodies to confirm subcellular distribution patterns.

• Autofluorescence mitigation: High-energy metabolic tissues often exhibit significant autofluorescence; use quenching agents such as Sudan Black B.

Research has shown that AK1 antibodies can effectively determine subcellular localization through immunofluorescence assays, revealing specific protein behaviors such as coacervation and formation of punctate microdomains in vivo .

How can I distinguish between AK1 and other adenylate kinase isoforms in my experiments?

Differentiating between AK1 and other adenylate kinase isoforms requires strategic experimental design:

• Isoform-specific antibodies: Select antibodies raised against unique epitopes not conserved across isoforms. Compare the immunogen sequence of candidate antibodies with all AK isoforms to predict potential cross-reactivity.

• Subcellular fractionation: Different AK isoforms have distinct subcellular localizations that can aid in identification:

  • AK1: Primarily cytosolic

  • AK2: Predominantly mitochondrial

• Knockout models: Utilize tissues/cells from AK1 knockout models to confirm antibody specificity against particular isoforms.

• Expression pattern analysis: Compare detection across tissues with known differential expression of AK isoforms.

• 2D gel electrophoresis: Separate isoforms by both isoelectric point and molecular weight before immunoblotting.

Research has demonstrated distinct localization patterns of AK1 and AK2 in specialized cells. For example, in spermatozoa, AK1 localizes to whole flagella while AK2 specifically localizes to midpieces, providing a means to distinguish between these isoforms .

What are optimal Western blotting protocols for detecting AK1 in tissues with variable expression levels?

Optimizing Western blotting protocols for AK1 detection across different tissue types requires:

• Sample preparation adjustments:

  • For high-expression tissues (heart, skeletal muscle): Use lower protein loading (10-15 μg)

  • For low-expression tissues: Increase loading to 25-30 μg

  • Include phosphatase inhibitors to preserve potentially important AK1 phosphorylation states

• Gel selection: 4-12% gradient gels provide optimal resolution for the 22 kDa AK1 protein.

• Transfer parameters: Use shorter transfer times (60-90 minutes) at lower voltage to prevent protein loss.

• Blocking optimization: 5% non-fat milk in PBST works effectively for most applications.

• Antibody dilutions:

  • Primary antibody: Start with manufacturer recommendations (typically 1:500-1:2000)

  • Secondary antibody: HRP-conjugated or fluorescently-labeled secondaries at 1:5000-1:10000

• Detection method selection: Both chemiluminescence and fluorescence-based detection systems work well; consider fluorescence-based systems for quantitative analysis.

Published protocols demonstrate effective AK1 detection using antibody dilutions of 1:1000 with secondary antibodies like goat anti-rabbit IgG conjugated to fluorescent markers .

How can I design co-immunoprecipitation experiments to study AK1 protein interactions?

For effective co-immunoprecipitation (co-IP) of AK1 and its interaction partners:

• Lysis buffer composition: Use mild non-ionic detergents (0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions. PBS with 0.02% sodium azide, 50% glycerol, pH 7.3 has been used successfully with AK1 antibodies .

• Antibody selection: Choose antibodies with good immunoprecipitation efficiency that don't interfere with protein interaction sites. Polyclonal antibodies often perform better for co-IP than monoclonals.

• Control selection:

  • IgG control IP to assess non-specific binding

  • Input samples to evaluate IP efficiency

  • Reverse IP (using antibodies against suspected interaction partners)

• Washing stringency: Balance between removing non-specific binding and preserving true interactions. Typically use 3-5 washes with lysis buffer containing reduced detergent.

• Elution strategy: Consider native elution with competing peptides for downstream functional assays.

Research has successfully employed co-IP techniques to demonstrate that soluble ODF4, a protein involved in sperm flagellar shape and movement, can be co-immunoprecipitated with AK1 and AK2 in wild-type spermatozoa, indicating functional interactions between these proteins .

What are common challenges when detecting AK1 in metabolically active tissues?

Detecting AK1 in metabolically active tissues presents several technical challenges:

• High background interference: Tissues like heart and skeletal muscle often exhibit high background due to endogenous peroxidases and biotin, which can mask specific AK1 signal.

• Rapid protein degradation: Metabolically active tissues require quick processing to prevent enzymatic degradation of target proteins.

• Post-translational modifications: Tissue-specific modifications of AK1 may affect antibody binding and detection sensitivity.

• Protein-protein interactions: AK1 associations with other proteins may mask epitopes, reducing antibody accessibility.

• Variable expression levels: High expression in some cells may mask detection in cells with lower expression within the same tissue.

Methodological solutions include:

• Extended blocking steps (1-2 hours) with 5-10% normal serum

• Hydrogen peroxide treatment to quench endogenous peroxidases

• Optimized antigen retrieval (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Signal amplification approaches for tissues with lower AK1 expression

Mouse heart and skeletal muscle have been identified as positive control tissues for AK1 antibody testing, as they express high levels of this protein .

How can I correlate AK1 protein levels with enzymatic activity in research samples?

To effectively correlate AK1 protein detection with enzymatic activity:

• Parallel analysis workflow:

  • Split samples for simultaneous protein quantification and activity assays

  • Standardize extraction conditions to preserve both protein integrity and enzymatic activity

  • Perform Western blotting with AK1 antibodies for protein quantification

  • Measure AK1 activity using coupled enzymatic assays that monitor ATP production

• Activity-to-protein ratio calculation: Normalize enzymatic activity measurements to AK1 protein levels determined by antibody detection.

• Inhibitor studies: Use AK1-specific inhibitors to confirm specificity of activity measurements relative to protein levels.

• Recombinant standards: Create calibration curves with purified AK1 for absolute quantification.

• Genetic manipulation models: Compare wild-type and AK1 overexpression/knockout models to establish relationship between protein levels and activity.

Research on cardiac-specific AK1 overexpressing mice (AK1-OE) has employed both protein detection and functional assays to correlate AK1 levels with enzymatic activity, demonstrating the value of this combined approach .

What considerations should be made when using AK1 antibodies in genetic knockout validation studies?

When designing knockout validation studies with AK1 antibodies:

• Experimental controls:

  • Include wild-type and AK1 knockout samples processed in parallel

  • Incorporate loading controls (GAPDH, β-actin) for normalization

  • Use multiple antibodies targeting different epitopes if available

• Genetic verification approach:

  • Confirm knockout status at DNA level using PCR (as described for AK1-OE mice using multiplex PCR protocols)

  • Verify transcript levels using qRT-PCR to detect both transgenic and endogenous AK1 mRNA

• Protein detection strategy:

  • Test antibodies at multiple dilutions to ensure optimal detection

  • Evaluate specificity across different applications (WB, IF, IHC)

  • Consider potential cross-reactivity with other adenylate kinase isoforms

• Compensatory mechanism assessment: Investigate whether other adenylate kinase isoforms are upregulated in AK1 knockout models.

• Functional correlation: Relate antibody detection results to phenotypic changes and adenylate kinase activity measurements.

Research has demonstrated the value of comprehensive validation approaches, showing that loss of AK1 antibody signal correlates with functional changes in models like AK1 knockout mice and cell lines .

How are AK1 antibodies being utilized to study the relationship between energy metabolism and disease pathology?

AK1 antibodies are enabling critical research at the intersection of energy metabolism and disease:

• Cardiac research applications:

  • Investigating AK1's role in protecting against ischemia/reperfusion injury

  • Studying how AK1 preserves ATP levels during ischemia

  • Examining AK1's contribution to activating AMPK during metabolic stress

  • Evaluating AK1 as a potential cardioprotective factor

• Reproductive biology:

  • Exploring AK1's association with sperm flagellar shape and movement

  • Investigating how AK1 interactions with proteins like ODF4 impact male fertility

  • Using AK1 antibodies to track subcellular localization in specialized reproductive cells

• Disease model investigations:

  • Employing AK1 antibodies to monitor expression changes in metabolic disorders

  • Correlating AK1 levels with disease progression and severity

• Therapeutic target assessment:

  • Using antibodies to validate AK1 as a potential therapeutic target

  • Monitoring AK1 expression changes in response to experimental treatments

Current research has demonstrated that AK1 plays subtle but important roles in maintaining normal basal metabolic function, with AK1 antibodies proving essential for tracking these relationships in both normal and pathological states .

What emerging techniques are enhancing the utility of AK1 antibodies in research?

Several emerging techniques are expanding the research applications of AK1 antibodies:

• Proximity ligation assays: Allowing visualization of protein-protein interactions involving AK1 in situ with single-molecule resolution.

• Super-resolution microscopy: Enabling detailed subcellular localization studies of AK1 beyond the diffraction limit of conventional microscopy.

• Single-cell analysis: Combining AK1 antibody detection with single-cell transcriptomics to correlate protein levels with gene expression profiles at the individual cell level.

• Mass cytometry (CyTOF): Integrating AK1 antibodies into multi-parameter panels for comprehensive phenotyping of metabolic states.

• CRISPR-based studies: Using genetically modified models in conjunction with AK1 antibodies to precisely define protein function.

• Tissue clearing techniques: Enabling 3D visualization of AK1 distribution across intact tissue specimens.

These advanced methods complement traditional antibody applications like Western blotting, immunohistochemistry, and ELISA, providing researchers with powerful new tools to investigate AK1 biology .

How do researchers address contradictory results when using different AK1 antibodies?

When faced with contradictory results from different AK1 antibodies, researchers should implement a systematic troubleshooting approach:

• Epitope mapping comparison:

  • Determine the specific regions recognized by each antibody

  • Assess whether differences in epitope recognition could explain conflicting results

  • Consider whether post-translational modifications might differentially affect epitope accessibility

• Validation method expansion:

  • Test antibodies using multiple techniques (WB, IP, IF, IHC)

  • Employ knockout/knockdown controls with each antibody

  • Use recombinant AK1 as a positive control

• Cross-reactivity assessment:

  • Test for potential cross-reactivity with other adenylate kinase isoforms

  • Evaluate species cross-reactivity if working with non-human samples

• Experimental condition standardization:

  • Ensure identical sample preparation methods across antibodies

  • Standardize protein denaturation conditions, as some epitopes may be conformation-dependent

  • Use consistent detection systems for all antibodies being compared

• Independent verification:

  • Employ non-antibody methods (mass spectrometry, activity assays) as orthogonal validation approaches

  • Consult published literature for previously reported discrepancies with specific antibodies

This comprehensive approach allows researchers to resolve contradictions and select the most appropriate antibodies for their specific experimental questions .