GAK Antibody, Biotin conjugated

What is GAK Antibody and what cellular processes does it help investigate?

GAK Antibody targets Cyclin G-associated kinase (GAK), a serine/threonine kinase that associates with cyclin G and CDK5. GAK functions as an auxilin homolog involved in the uncoating of clathrin-coated vesicles by Hsc70 in non-neuronal cells . This antibody enables researchers to investigate several critical cellular processes:

• Clathrin-mediated endocytosis and intracellular trafficking

• Dynamics of clathrin assembly and disassembly

• Cell cycle regulation (GAK expression oscillates during the cell cycle, peaking at G1)

• Receptor trafficking and signaling pathways

The antibody provides a valuable tool for studying these fundamental cellular mechanisms in non-neuronal cells, particularly in contexts where vesicular trafficking may be altered.

What are the key applications of biotin-conjugated GAK antibody?

The biotin-conjugated GAK antibody has several research applications, with ELISA being the most consistently reported application across various sources . The biotin conjugation provides specific advantages:

• ELISA (Enzyme-Linked Immunosorbent Assay): The primary validated application for quantitative detection and analysis of GAK protein

• Potential for Immunohistochemistry: While not explicitly validated for all biotin-conjugated forms, some GAK antibodies are suitable for IHC applications

• Immunofluorescence studies: The biotin tag enables secondary detection systems in microscopy applications

• Pull-down assays: Leveraging the strong biotin-streptavidin interaction for protein isolation

The biotin conjugation enhances detection sensitivity through amplification methods using streptavidin-based detection systems, making it particularly useful for detecting low abundance proteins in complex samples.

What is the molecular structure and characteristics of the target protein GAK?

GAK (Cyclin G-associated kinase) is a multidomain protein with several key structural characteristics:

• Molecular Weight: 143-150 kDa observed in Western blotting

• Domains: Contains a kinase domain (N-terminal) that distinguishes it from auxilin I, which lacks this domain

• Homology: High sequence homology with auxilin outside its kinase domain

• Classification: Belongs to the NAK (numb-associated kinase) family within the protein kinase superfamily

• UniProt ID: O14976 (human)

The crystal structure of the GAK catalytic domain has been determined both alone and in complex with specific single-chain antibodies (nanobodies), revealing an unusual activation segment interaction in its dimeric inactive state .

How should researchers prepare and store the GAK Antibody, Biotin conjugated?

Proper handling of GAK Antibody, Biotin conjugated is crucial for maintaining its activity and specificity:

Storage recommendations:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles that can compromise antibody integrity

• Some formulations contain 50% glycerol, allowing storage at -20°C without frequent freezing/thawing

Buffer composition:

• Typical storage buffer contains: 0.03% Proclin 300 as a preservative, 50% Glycerol, 0.01M PBS, pH 7.4

• Some preparations may contain additional stabilizers like BSA

Working dilutions:

• For ELISA applications, optimal dilutions should be determined experimentally

• For other potential applications like Western blotting or immunohistochemistry, dilutions of 1:500-1:1000 are typically recommended for similar GAK antibodies

How does the structure of GAK's catalytic domain influence antibody selection for specific functional studies?

The unique structural features of GAK's catalytic domain have significant implications for antibody selection in functional studies:

GAK exhibits unusually high plasticity in its catalytic domain, making it a frequent "off-target" of clinical kinase inhibitors . When designing experiments to study GAK function:

• Consider epitope location: Antibodies targeting different domains (kinase domain vs. auxilin-homologous regions) may reveal different functional aspects of GAK

• Structural conformations: GAK can exist in both monomeric and dimeric arrangements with distinct activation states

• Domain-specific interactions: The crystal structure reveals GAK can be trapped in different conformational states by specific nanobodies, suggesting antibodies may differentially detect active versus inactive GAK

Research by Zhang et al. has shown that co-crystallization with nanobody NbGAK_4 trapped GAK in a dimeric arrangement similar to the apo structure, whereas NbGAK_1 captured the activation segment of monomeric GAK in a well-ordered conformation representing features of the active kinase . This structural plasticity means researchers should carefully select antibodies based on which conformational state they wish to detect.

What controls and validation steps are essential when using GAK Antibody, Biotin conjugated in ELISA experiments?

Rigorous controls and validation are critical for generating reliable data with GAK Antibody, Biotin conjugated in ELISA:

Essential controls:

• Positive control: Use known GAK-expressing cell lysates (e.g., 293 cells)

• Negative control: Include samples with GAK knockdown or from tissues not expressing GAK

• Peptide competition assay: Preincubate antibody with immunizing peptide to confirm specificity

• Isotype control: Include matched rabbit IgG to assess non-specific binding

Validation steps:

• Cross-reactivity testing: Verify reactivity with human samples as indicated in product information

• Dilution optimization: Test serial dilutions to determine optimal antibody concentration

• Signal-to-noise ratio assessment: Evaluate background signal compared to specific signal

• Comparison with non-conjugated antibody: If available, compare results with non-biotinylated version to assess conjugation effects

When analyzing data, Western blot validation showing a single band at approximately 143-150 kDa can provide additional confidence in antibody specificity before proceeding with quantitative ELISA studies .

How can GAK Antibody be employed to study clathrin-mediated endocytosis and receptor trafficking pathways?

GAK plays a crucial role in clathrin-mediated endocytosis and receptor trafficking, making GAK antibodies valuable tools for investigating these processes:

Methodological approaches:

• Co-localization studies: Combine GAK antibody with clathrin markers to visualize uncoating processes in fixed cells

• Receptor trafficking analysis: Monitor changes in receptor distribution following GAK knockdown to identify GAK-dependent trafficking steps

• Pulse-chase experiments: Track internalized receptors in GAK-depleted versus normal cells

• Kinase activity assays: Use phospho-specific antibodies alongside GAK antibody to correlate kinase activity with trafficking events

Research insights:
Studies using GAK antibodies have revealed that GAK down-regulation significantly alters EGF receptor trafficking, resulting in receptor persistence in altered cellular compartments and dramatically increased receptor expression and tyrosine kinase activity (>50-fold) . This demonstrates how GAK antibodies can uncover critical regulatory mechanisms in receptor endocytosis and signaling.

For experimental design, researchers should consider that GAK functions downstream of dynamin in the endocytic pathway , and its effects on receptor trafficking may vary depending on receptor type and cell context.

What are the technical considerations when comparing results from different GAK antibody clones or conjugates?

When comparing results obtained using different GAK antibody preparations, researchers should consider several technical factors:

Key considerations:

• Epitope differences: Different antibodies target distinct regions of GAK (N-terminal vs. C-terminal), affecting detection of splice variants or post-translationally modified forms

• Immunogen variation: Antibodies generated against synthetic peptides vs. recombinant proteins may have different specificities

• Conjugation effects: Biotin conjugation may affect antibody binding characteristics compared to unconjugated versions

• Species reactivity: Verify cross-reactivity profiles, as some GAK antibodies react with human only, while others detect mouse and rat homologs

Comparative analysis approach:

• Document all antibody characteristics (catalog numbers, hosts, epitopes, conjugates)

• Run parallel experiments with standardized protocols

• Include internal controls recognizable by all antibodies being compared

• Consider using antibody mixtures for comprehensive detection if studying complex samples

The crystallographic studies of GAK reveal significant conformational flexibility , suggesting different antibodies may preferentially recognize specific structural states of the protein.

How can researchers leverage GAK antibodies to investigate the relationship between clathrin-mediated endocytosis and cell cycle regulation?

GAK expression oscillates during the cell cycle (peaking at G1) , suggesting an interplay between endocytic trafficking and cell cycle progression that can be explored using GAK antibodies:

Experimental strategies:

• Cell synchronization: Combine with GAK immunostaining to track expression and localization changes throughout cell cycle phases

• Dual-labeling approaches: Co-stain for GAK and cell cycle markers (cyclins, CDKs) to establish temporal relationships

• Quantitative analysis: Use flow cytometry with GAK antibody staining to correlate GAK levels with DNA content

• Perturbation studies: Compare endocytic rates in different cell cycle phases using GAK as a marker for clathrin uncoating machinery

Research applications:
Studies have shown that down-regulation of GAK can have profound biological consequences, including altered growth properties. In CV1P cells, GAK down-regulation resulted in outgrowth of cells in soft agar, suggesting potential links to tumorigenesis . This indicates GAK antibodies could be valuable tools for investigating how dysregulation of endocytic processes contributes to cancer development.

Table 1: GAK Antibody, Biotin conjugated - Key Specifications

Table 2: GAK Protein Characteristics