AK7 Antibody

What is AK7 and what is its physiological significance in research?

Adenylate kinase 7 (AK7) is a 723 amino acid enzyme with a molecular weight of approximately 82.7 kDa that plays a crucial role in cellular energy homeostasis. It catalyzes the conversion of one adenosine triphosphate (ATP) and one adenosine monophosphate (AMP) into two adenosine diphosphate (ADP) molecules .

This enzymatic activity is vital for maintaining the balance of adenine nucleotides within cells, which influences numerous metabolic processes and cellular signaling pathways. AK7's physiological significance stems from:

• Its subcellular localization in the cytoplasm

• Expression patterns primarily in sperm and airway epithelial cells

• Involvement in protein phosphorylation pathways

• Association with the Adenylate kinase protein family and Dpy-30 protein family

Research into AK7 has potential implications for understanding conditions associated with chromosome 14, including Alzheimer's disease and a1-antitrypsin deficiency .

What applications are AK7 antibodies optimized for in experimental protocols?

AK7 antibodies have been validated for multiple research applications, each requiring specific optimization strategies:

When designing experiments, consider starting with the manufacturer's recommended dilutions and optimize based on your specific experimental conditions and tissue/cell types .

How should I select between polyclonal and monoclonal AK7 antibodies for specific research applications?

The choice between polyclonal and monoclonal AK7 antibodies depends on your experimental goals and requirements:

Polyclonal AK7 Antibodies:

• Recognize multiple epitopes on the AK7 protein

• Often provide stronger signal due to multiple binding sites

• Better for detecting denatured proteins in Western blots

• Examples include rabbit polyclonal antibodies targeting specific regions like the C-terminal domain (629-656 amino acids)

• Ideal for initial characterization studies or when protein expression is low

Monoclonal AK7 Antibodies:

• Recognize a single epitope with high specificity

• Provide consistent lot-to-lot reproducibility

• Reduced background and cross-reactivity

• Examples include mouse monoclonal IgG1 kappa light chain antibodies like AK7 (D-5)

• Optimal for applications requiring high specificity like immunoprecipitation

For quantitative studies or when comparing expression across multiple samples, monoclonal antibodies often provide more consistent results. For protein detection in complex samples or denatured conditions, polyclonal antibodies may offer greater sensitivity .

What validation methods should I employ to confirm AK7 antibody specificity?

Thorough validation is critical for ensuring reliable research results with AK7 antibodies:

• Protein Array Testing: Validate against target protein plus other non-specific proteins (e.g., 383 other proteins) to confirm specificity

• Orthogonal RNA-seq Validation: Compare antibody-based protein detection with mRNA expression data to ensure correlation

• Knockout/Knockdown Controls: Test antibody in samples where AK7 has been knocked out or knocked down through siRNA/CRISPR

• Western Blot Analysis: Confirm detection of a single band at the expected molecular weight (~83 kDa)

• Peptide Competition Assay: Pre-incubate antibody with the immunogen peptide to demonstrate signal reduction

• Multi-species Reactivity Testing: Confirm expected cross-reactivity with human, mouse, rat or other relevant species based on sequence homology

• Cross-validation: Compare results using multiple antibodies targeting different epitopes of AK7

Manufacturers often perform initial validation testing, such as Boster Bio validating all antibodies on WB, IHC, ICC, Immunofluorescence, and ELISA with known positive and negative controls .

What are optimal storage and handling practices for preserving AK7 antibody activity?

Proper storage and handling are essential for maintaining antibody performance throughout your research project:

Storage Conditions:

• Store at -20°C for long-term storage (stable up to one year)

• Store at 4°C for short-term storage (up to three months)

• Antibodies are typically supplied in PBS (pH 7.2) with 40-50% glycerol and preservatives like 0.02% sodium azide

Handling Best Practices:

• Avoid repeated freeze-thaw cycles which can degrade antibody activity

• Aliquot antibodies upon receipt to minimize freeze-thaw events

• Allow antibodies to equilibrate to room temperature before opening

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

• Use sterile techniques when handling to prevent contamination

• Return to recommended storage temperature immediately after use

Working Solution Preparation:

• Dilute in appropriate buffer just before use

• Discard unused diluted antibody rather than storing

• For dilution, use high-quality, filtered buffer solutions

Following these practices will help ensure consistent experimental results and maximize the lifespan of your AK7 antibody investment .

How can I troubleshoot non-specific binding or weak signal issues with AK7 antibodies?

When encountering problems with AK7 antibody performance, systematic troubleshooting can help identify and resolve issues:

For Non-specific Binding:

• Increase blocking time/concentration (try 5% BSA or 5% non-fat dry milk)

• Optimize antibody dilution (test serial dilutions)

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

• Pre-adsorb antibody with tissues/cells lacking AK7

• Increase washing duration and frequency

• Use more stringent washing buffer

• For polyclonal antibodies, consider affinity purification against the immunogen

For Weak Signal:

• Increase antibody concentration

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

• Optimize antigen retrieval methods for IHC applications

• Use signal enhancement systems (e.g., biotin-streptavidin amplification)

• Ensure target protein is not degraded (add protease inhibitors)

• Verify sample preparation maintains native protein conformation

• Try different detection methods (e.g., switch from HRP to more sensitive fluorescent detection)

For Western Blot Specific Issues:

• Optimize transfer conditions (time, voltage, buffer composition)

• Ensure adequate protein loading (typically 20-50 μg total protein)

• Verify protein molecular weight (AK7 should appear at ~83 kDa)

• Try reducing or non-reducing conditions based on antibody specifications

Document all troubleshooting steps and results to develop an optimized protocol for your specific experimental conditions .

What epitope considerations are important when selecting AK7 antibodies for specific applications?

The epitope recognized by an AK7 antibody significantly impacts its performance in different applications:

Known AK7 Epitope Regions:

• C-terminal region: amino acids 629-656

• Key immunogenic sequence: QAKDLFNQEDEEEEDDVRGRMFPFDKLIIPEFVCALDASDEFLKERVINLPESIVAGTHYSQDRFLRALSNYRDINIDDETVFNYFDELEIHPIHIDVGKLEDAQNRLAIKQLIKEIGEPRNYGLTDEEKAEEE

Application-Specific Considerations:

• For Western Blotting:

  • Linear epitopes are preferable as proteins are denatured

  • Antibodies recognizing C-terminal regions often perform well

  • Consider whether post-translational modifications might mask epitopes

• For Immunoprecipitation:

  • Conformational epitopes on surface-exposed regions work best

  • Avoid epitopes that might be involved in protein-protein interactions

• For Immunohistochemistry:

  • Consider fixation effects on epitope accessibility

  • Some epitopes may be masked by formalin cross-linking

  • Test multiple antibodies targeting different regions

• For Flow Cytometry:

  • Select antibodies targeting extracellular domains if AK7 is membrane-associated

  • Consider whether epitopes are accessible in native conformation

When possible, review published literature using specific AK7 antibody clones to assess performance in your application of interest. Selecting an antibody with an epitope appropriate for your experimental conditions will significantly improve results .

How do species cross-reactivity profiles influence AK7 antibody selection for comparative studies?

Understanding species cross-reactivity is crucial when designing comparative studies across different model organisms:

Cross-Reactivity Profiles of Common AK7 Antibodies:

Considerations for Cross-Species Studies:

• Sequence Homology Analysis:

  • Verify sequence conservation of the epitope region across species

  • Higher homology in the epitope region predicts better cross-reactivity

• Validation Requirements:

  • Always validate antibodies in each species independently

  • Do not assume reactivity based on manufacturer claims alone

  • Include positive and negative control tissues from each species

• Application Differences:

  • An antibody may cross-react in one application (e.g., WB) but not another (e.g., IHC)

  • Different fixation methods may affect cross-reactivity in IHC applications

• Alternative Approaches:

  • Consider species-specific antibodies when possible

  • For novel species, custom antibody development may be necessary

When conducting evolutionary or comparative studies, carefully document antibody performance across species to ensure valid cross-species comparisons .

What are the advantages and limitations of different conjugated AK7 antibodies for multiplex immunofluorescence studies?

Conjugated AK7 antibodies enable multiplex studies, each with specific advantages and limitations:

Available Conjugations for AK7 Antibodies:

Multiplex Strategy Considerations:

• Spectral Compatibility:

  • Select fluorophores with minimal spectral overlap

  • Consider your instrument's laser lines and filter sets

  • Use spectral unmixing for closely overlapping fluorophores

• Primary Antibody Host Species:

  • Choose primary antibodies from different host species to avoid cross-reactivity

  • AK7 antibodies are available in rabbit and mouse hosts

• Signal Intensity Balancing:

  • Match brighter fluorophores with lower-expressed targets

  • Adjust antibody concentrations to balance signal intensities

• Potential Pitfalls:

  • Fluorophore conjugation may affect antibody affinity

  • Larger fluorophores might cause steric hindrance

  • Direct conjugates eliminate secondary cross-reactivity but may have lower sensitivity

When designing multiplex experiments, conduct single-stain controls to verify specificity and performance of each conjugated antibody independently before combining them .

How can I design experiments to study AK7's role in cellular energy homeostasis?

To investigate AK7's function in energy metabolism, consider these experimental approaches:

Experimental Strategies:

• Expression Manipulation Studies:

  • Overexpression using expression vectors with tagged AK7

  • Knockdown using siRNA or shRNA targeting AK7

  • CRISPR/Cas9 gene editing to create knockout models

  • Compare adenylate kinase activity in manipulated vs. control cells

• Metabolic Analysis:

  • Measure ATP:ADP:AMP ratios using luminescence-based assays

  • Monitor cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

  • Assess mitochondrial function in cells with altered AK7 expression

  • Track metabolic pathway flux using isotope-labeled substrates

• Protein Interaction Studies:

  • Co-immunoprecipitation with AK7 antibodies to identify binding partners

  • Proximity labeling techniques (BioID, APEX) to map AK7 protein interaction network

  • Yeast two-hybrid screening for novel interactors

• Subcellular Localization:

  • Immunofluorescence with AK7 antibodies to determine cytoplasmic distribution

  • Co-localization with metabolic enzymes or subcellular markers

  • Live-cell imaging using fluorescently tagged AK7

• Physiological Response Assays:

  • Examine AK7 expression and activity under energy stress conditions

  • Assess cellular responses to metabolic inhibitors in AK7-modulated cells

  • Investigate tissue-specific functions in sperm and airway epithelial cells

Key Controls and Considerations:

• Include both gain- and loss-of-function approaches

• Validate antibody specificity before conducting critical experiments

• Select appropriate cell types (those naturally expressing AK7)

• Consider potential compensation by other adenylate kinase isoforms

This comprehensive approach will provide insights into AK7's specific contribution to cellular energy homeostasis beyond what is currently known about its enzymatic function .

What methodological approaches can address potential discrepancies between AK7 antibody-based protein detection and mRNA expression data?

When protein and mRNA data for AK7 don't align, systematic investigation is needed:

Causes of Protein-mRNA Discrepancies:

• Post-transcriptional Regulation:

  • miRNA-mediated repression of AK7 translation

  • RNA binding proteins affecting mRNA stability or translation efficiency

  • Alternative splicing creating protein variants not detected by certain antibodies

• Post-translational Regulation:

  • Protein degradation rates differing from mRNA turnover

  • Stimulus-dependent protein stabilization or degradation

  • Subcellular compartmentalization affecting antibody accessibility

• Technical Limitations:

  • Antibody specificity issues or epitope masking

  • RNA quantification method sensitivity differences

  • Sample preparation differences between protein and RNA experiments

Resolution Strategies:

• Orthogonal Validation Approaches:

  • Use multiple antibodies targeting different AK7 epitopes

  • Employ mass spectrometry-based proteomics for antibody-independent detection

  • Compare with enhanced RNA-seq techniques that capture all transcript isoforms

• Time-course Experiments:

  • Monitor both mRNA and protein levels over time following stimulation

  • Account for temporal delays between transcription and translation

• Intervention Studies:

  • Use proteasome inhibitors to assess protein degradation contributions

  • Apply translation inhibitors to measure protein half-life

  • Target specific RNA regulatory elements using CRISPR techniques

• Isoform-specific Analysis:

  • Design PCR primers or RNA-seq analysis to detect specific transcript variants

  • Use antibodies targeting conserved regions versus isoform-specific regions

  • Perform Western blots to detect multiple protein bands representing isoforms

When planning experiments, incorporate these approaches to ensure comprehensive characterization of AK7 expression and function, particularly in tissues where post-transcriptional regulation may be significant .

How should I interpret AK7 immunohistochemistry results in the context of tissue-specific expression patterns?

Accurate interpretation of AK7 immunohistochemistry requires understanding its established expression patterns and technical considerations:

Known AK7 Expression Patterns:

• Primarily expressed in sperm and airway epithelial cells

• Localized to the cytoplasm

• May have tissue-specific isoforms or expression levels

Interpretation Guidelines:

• Staining Pattern Analysis:

  • Cytoplasmic staining consistent with AK7's known localization

  • Assess staining intensity variations across cell types within a tissue

  • Evaluate subcellular distribution (diffuse vs. punctate patterns)

  • Compare with published Human Protein Atlas data when available

• Quantification Approaches:

  • Use digital image analysis for objective quantification

  • Score based on staining intensity (0-3+) and percentage of positive cells

  • Apply H-score or Allred scoring systems for semi-quantitative assessment

• Critical Controls:

  • Positive control tissues (airway epithelial cells)

  • Negative control tissues (tissues known not to express AK7)

  • Isotype controls to assess non-specific binding

  • Peptide competition controls to confirm specificity

• Potential Artifacts and Pitfalls:

  • Edge artifacts at tissue margins

  • Necrotic tissue showing non-specific staining

  • Endogenous peroxidase activity if using HRP detection

  • Epitope masking due to fixation

  • Cross-reactivity with similar proteins

• Comparative Analysis:

  • Compare results across multiple antibodies when possible

  • Correlate with functional data or disease parameters

  • Consider developmental or disease-related changes in expression

For research involving novel tissues or disease states, validate findings using complementary techniques such as in situ hybridization or RT-PCR to confirm AK7 expression patterns .

What experimental design considerations are important when using AK7 antibodies to study disease-associated mechanisms?

When investigating AK7 in disease contexts, comprehensive experimental design is crucial:

Disease-Relevant Considerations:

• Connection to Known Pathologies:

  • AK7 gene is located on chromosome 14, associated with Alzheimer's disease and a1-antitrypsin deficiency

  • Consider potential roles in cellular energy metabolism disorders

  • Investigate relationships to cilia-related diseases given expression in airway epithelial cells

• Sample Selection and Controls:

  • Include age-matched and sex-matched controls

  • Consider disease stage progression (early, middle, late)

  • Use appropriate disease models (animal models, patient-derived cells)

  • Include both affected and unaffected tissues from the same patient when possible

• Technical Approaches:

  For Expression Analysis:

  • Quantitative Western blotting with normalizing controls

  • Immunohistochemistry with digital quantification

  • Flow cytometry for cell-specific expression in mixed populations

  For Functional Studies:

  • Activity assays measuring adenylate kinase function

  • Metabolic flux analysis in disease vs. normal states

  • Genetic rescue experiments in disease models

• Correlation with Clinical Parameters:

  • Relate AK7 expression/activity to disease severity markers

  • Longitudinal sampling when possible

  • Stratify analysis based on patient subgroups or treatment responses

• Alternative Splicing and Isoforms:

  • Investigate disease-specific isoform expression

  • Use antibodies that can distinguish between isoforms

  • Complement with RNA-seq to detect splice variants

Validation Strategies:

• Use multiple methodologies to confirm findings (protein, mRNA, activity)

• Include independent cohorts for replication

• Perform in vitro functional validation of mechanisms

• Consider translational relevance through correlation with clinical outcomes

This comprehensive approach ensures robust findings when studying AK7's potential roles in disease processes and may reveal novel therapeutic targets .

How can I optimize protocols for co-immunoprecipitation experiments using AK7 antibodies?

Successful co-immunoprecipitation (co-IP) with AK7 antibodies requires careful optimization:

Protocol Optimization Steps:

• Antibody Selection:

  • Choose antibodies specifically validated for IP applications

  • The AK7 (D-5) antibody has been validated for immunoprecipitation

  • Consider using agarose-conjugated AK7 antibodies for direct IP

  • Avoid antibodies whose epitopes might be involved in protein interactions

• Lysis Buffer Optimization:

  • Start with non-denaturing buffers (e.g., RIPA or NP-40-based)

  • Adjust salt concentration (150-500 mM) to balance specificity and yield

  • Include protease and phosphatase inhibitors

  • Consider detergent type and concentration based on subcellular localization

  • For cytoplasmic AK7, mild detergents like 0.5% NP-40 or 1% Triton X-100 are suitable

• IP Conditions:

  • Pre-clear lysates with control IgG/Protein A/G to reduce non-specific binding

  • Optimize antibody amount (typically 2-5 μg per reaction)

  • Test various incubation times (2 hours vs. overnight at 4°C)

  • Include gentle rotation to maintain antibody suspension without damaging complexes

• Washing Stringency:

  • Balance between maintaining specific interactions and reducing background

  • Test increasing salt concentrations in wash buffers (150-500 mM)

  • Optimize number of washes (typically 3-5)

  • Consider adding low concentrations of detergent to wash buffers

• Elution Methods:

  • Gentle elution with low pH glycine buffer for reversible interactions

  • Direct boiling in SDS sample buffer for stronger detection of interactions

  • Peptide competition elution for higher specificity

Critical Controls:

• Input control (5-10% of starting material)

• IgG isotype control IP processed identically

• Reciprocal IP with antibodies against suspected interacting partners

• IP in cells with AK7 knockdown or knockout as negative control

Detection Methods:

• Western blot with antibodies against suspected interacting partners

• Mass spectrometry for unbiased identification of interaction partners

• Consider crosslinking approaches for transient interactions

Successful co-IP can reveal AK7's protein interaction network, providing insights into its functional roles beyond enzymatic activity .

What considerations are important when designing AK7 antibody-based flow cytometry experiments?

While AK7 is primarily a cytoplasmic protein, flow cytometry can be valuable for studying its expression in specific cell populations with proper protocol design:

Key Protocol Considerations:

• Cell Preparation and Fixation:

  • For intracellular AK7, permeabilization is essential

  • Test different fixatives (4% paraformaldehyde vs. methanol)

  • Optimize permeabilization agents (0.1-0.5% saponin, 0.1% Triton X-100, or commercial permeabilization buffers)

  • Maintain cell integrity while ensuring antibody access to cytoplasmic compartments

• Antibody Selection:

  • Choose flow cytometry-validated AK7 antibodies

  • Consider fluorochrome brightness based on expected expression levels

  • Available conjugates include FITC, PE, and mFluor Violet 450

  • For multicolor panels, select fluorochromes with minimal spectral overlap

• Protocol Optimization:

  • Titrate antibody concentration to determine optimal signal-to-noise ratio

  • Test different incubation times and temperatures

  • Include blocking steps to reduce non-specific binding

  • Optimize washing steps to reduce background

• Panel Design for Co-expression Studies:

  • Include lineage markers to identify specific cell populations

  • Add functional markers relevant to your research question

  • Consider compensation requirements when selecting fluorochromes

  • Limit panel to capabilities of your flow cytometer

• Controls:

  • Unstained cells for autofluorescence assessment

  • Single-color controls for compensation

  • Fluorescence minus one (FMO) controls

  • Isotype controls matched to antibody class and fluorochrome

  • Positive and negative cell populations (when known)

• Analysis Considerations:

  • Gate strategy should include doublet discrimination and viability selection

  • Assess both percentage of positive cells and mean fluorescence intensity

  • Consider using histogram overlays to visualize shifts in expression

  • For heterogeneous populations, look for discrete positive populations