ADCK5 Antibody

What is ADCK5 and what are its key characteristics in humans?

ADCK5 (aarF domain containing kinase 5) is a membrane-localized protein with significant roles in protein phosphorylation pathways. In humans, the canonical ADCK5 protein consists of 580 amino acid residues with a molecular mass of approximately 65.8 kDa . It belongs to the ADCK protein kinase family and is widely expressed across numerous tissue types, making it an important target for various research applications .

The protein is also known by the synonym "uncharacterized aarF domain-containing protein kinase 5" and has documented orthologs in multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken . This evolutionary conservation suggests functionally significant roles across vertebrate species.

Which experimental applications are most suitable for ADCK5 antibody research?

When designing experiments, researchers should consider that ADCK5's membrane localization may require specific sample preparation techniques to ensure optimal antibody accessibility .

How should researchers select an appropriate ADCK5 antibody for their experimental system?

Selection criteria should include:

• Target epitope location: N-terminal vs. C-terminal vs. middle region antibodies may yield different results depending on protein interactions or post-translational modifications .

• Species reactivity: Verify cross-reactivity with your experimental model organism. Many available antibodies react with human ADCK5, while others demonstrate reactivity with mouse or rat orthologs .

• Clonality considerations: Polyclonal antibodies may provide higher sensitivity but potentially lower specificity compared to monoclonal options.

• Validated applications: Ensure the antibody has been specifically validated for your intended application (WB, ELISA, IF, IHC) through manufacturer documentation or literature citations .

• Conjugation requirements: Determine whether unconjugated antibodies are sufficient or if conjugated versions (biotin, FITC, HRP, etc.) would benefit your experimental design .

What are the optimal protocols for validating ADCK5 antibody specificity?

A rigorous validation approach should incorporate multiple methods:

• Positive and negative controls: Utilize tissues or cell lines with known ADCK5 expression levels. Consider knockout or knockdown models as negative controls.

• Pre-absorption testing: Pre-incubate antibody with purified ADCK5 protein prior to application, which should eliminate specific staining.

• Multiple antibody comparison: Employ antibodies targeting different epitopes of ADCK5 to confirm consistent detection patterns.

• Molecular weight verification: Confirm detection at the expected 65.8 kDa molecular weight in Western blot applications .

• Cross-reactivity assessment: Evaluate potential cross-reactivity with other ADCK family members, particularly in systems where multiple family members are expressed.

How can researchers optimize ADCK5 detection in immunohistochemistry across different tissue types?

Successful immunohistochemical detection of ADCK5 requires tailored approaches:

• Fixation optimization: Compare paraformaldehyde, formalin, and alcohol-based fixatives to determine optimal epitope preservation.

• Antigen retrieval techniques:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) often provides good results for membrane proteins

  • Enzymatic retrieval using proteinase K may be necessary for heavily fixed tissues

  • Test both methods to determine optimal conditions for ADCK5 detection

• Blocking parameters: Given ADCK5's widespread expression, optimizing blocking conditions is critical to reduce background:

  • Use 3-5% normal serum from the species in which the secondary antibody was raised

  • Include 0.1-0.3% Triton X-100 for membrane permeabilization

  • Consider adding 1% BSA to reduce non-specific binding

• Signal amplification: For tissues with lower ADCK5 expression, consider biotin-streptavidin amplification systems or tyramide signal amplification .

What experimental design considerations are important when studying ADCK5 protein interactions?

When investigating ADCK5 interactions, consider these methodological approaches:

• Co-immunoprecipitation optimization:

  • Use membrane-compatible lysis buffers containing 0.5-1% NP-40 or Triton X-100

  • Include phosphatase inhibitors to preserve potential phosphorylation-dependent interactions

  • Consider crosslinking approaches for transient interactions

• Proximity labeling techniques:

  • BioID or APEX2 fusion constructs can reveal the proximal interactome of membrane-localized ADCK5

  • Design constructs carefully to avoid disrupting membrane localization

• Mass spectrometry sample preparation:

  • Implement specialized membrane protein extraction protocols

  • Consider sequential extraction methods to separate cytosolic from membrane-bound interaction partners

• Controls for specificity:

  • Include IgG controls matched to the host species of your ADCK5 antibody

  • Use ADCK5-depleted samples as negative controls

How does ADCK5 antibody performance vary across different experimental applications?

Antibody performance varies considerably across applications, and researchers should consider:

Researchers should conduct pilot studies with positive controls to optimize conditions for each specific application .

How can researchers address non-specific binding when using ADCK5 antibodies?

Non-specific binding is a common challenge when working with membrane protein antibodies:

• Increase blocking stringency:

  • Extend blocking time to 2 hours at room temperature

  • Test alternative blocking agents (milk vs. BSA vs. serum)

  • Consider commercial blocking buffers specifically designed for membrane proteins

• Optimize antibody concentration:

  • Perform titration experiments to determine minimal effective concentration

  • Extended incubation at 4°C with lower antibody concentration may improve signal-to-noise ratio

• Modify washing protocols:

  • Increase washing duration and/or number of washes

  • Add low concentrations of detergent (0.05-0.1% Tween-20) to wash buffers

• Pre-adsorption strategies:

  • Pre-incubate antibody with tissues/cells known to lack ADCK5 expression

  • For polyclonal antibodies, consider affinity purification against the immunizing peptide

What are the methodological considerations for detecting post-translational modifications of ADCK5?

Since ADCK5 is involved in protein phosphorylation pathways, studying its own modifications requires:

• Phosphorylation-specific approaches:

  • Use phosphatase inhibitors in all extraction buffers

  • Consider phosphorylation-specific antibodies if available

  • Implement Phos-tag SDS-PAGE to separate phosphorylated from non-phosphorylated species

• Sample preparation optimization:

  • Rapid sample processing minimizes phosphatase activity

  • Use caution with heat denaturation, which may affect phosphorylation detection

  • Consider kinase activation or inhibition treatments to validate phosphorylation sites

• Mass spectrometry approaches:

  • Enrich for phosphopeptides using titanium dioxide or IMAC

  • Implement parallel reaction monitoring for targeted detection of predicted modification sites

  • Use isotope labeling to compare modification states between experimental conditions

How can multiplexed detection systems be implemented for studying ADCK5 in complex cellular contexts?

Multiplexed approaches enable simultaneous detection of ADCK5 with interacting partners or pathway components:

• Multi-color immunofluorescence:

  • Select primary antibodies from different host species

  • Use highly cross-absorbed secondary antibodies to prevent cross-reactivity

  • Implement spectral unmixing for channels with potential overlap

• Sequential immunoblotting:

  • Carefully optimize stripping conditions to remove previous antibodies without affecting membrane-bound proteins

  • Consider using different detection methods (chemiluminescence vs. fluorescence) for multiplexing

• Multiplex flow cytometry:

  • Utilize directly conjugated antibodies with non-overlapping fluorophores

  • Include appropriate compensation controls

  • Consider permeabilization optimization for this membrane protein

What quantitative analysis approaches are most appropriate for ADCK5 expression studies?

For accurate quantification of ADCK5 levels:

• Western blot densitometry considerations:

  • Use loading controls appropriate for membrane proteins (Na⁺/K⁺-ATPase, cadherin)

  • Establish linear range of detection with standard curves

  • Apply statistical approaches appropriate for ratio data

• qPCR method optimization:

  • Design primers spanning exon junctions to avoid genomic DNA amplification

  • Validate primer efficiency using standard curves

  • Select reference genes with stable expression across your experimental conditions

• Mass spectrometry quantification:

  • Implement labeled reference peptides for absolute quantification

  • Consider parallel reaction monitoring for improved sensitivity

  • Develop extraction methods optimized for membrane proteins

How can ADCK5 antibodies be utilized in single-cell analysis approaches?

Adapting ADCK5 detection to single-cell methodologies requires:

• Single-cell immunofluorescence optimization:

  • Titrate antibody concentration to minimize background

  • Implement image analysis algorithms to quantify membrane localization

  • Consider super-resolution microscopy techniques for detailed localization studies

• Mass cytometry (CyTOF) adaptation:

  • Metal-conjugated antibodies require validation for epitope accessibility

  • Optimize cell fixation and permeabilization for membrane protein detection

  • Develop analysis pipelines that account for ADCK5's expression patterns

• Single-cell Western approaches:

  • Modify lysis conditions for efficient release of membrane-bound ADCK5

  • Optimize protein separation parameters based on ADCK5's molecular weight

  • Validate detection sensitivity with control cell populations

What considerations are important when designing CRISPR/Cas9 experiments to study ADCK5 function?

When implementing genome editing approaches:

• Guide RNA design strategies:

  • Target functional domains predicted from sequence analysis

  • Avoid regions with known splice variants to ensure complete knockout

  • Consider targeting conserved regions for cross-species studies

• Validation methodology:

  • Implement Western blot with antibodies targeting different epitopes to confirm knockout

  • Design PCR strategies to verify genomic modifications

  • Sequence the targeted region to confirm precise editing

• Functional rescue experiments:

  • Design expression constructs resistant to guide RNA targeting

  • Consider tagged versions for distinguishing endogenous from exogenous protein

  • Implement inducible expression systems to control rescue timing