GAK Antibody

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

GAK (Cyclin-G-associated kinase) is a multifunctional protein that associates with cyclin G and CDK5. It functions as an auxilin homolog involved in uncoating clathrin-coated vesicles by Hsc70 in non-neuronal cells. Its expression pattern oscillates slightly during the cell cycle, with peak expression in G1 phase . GAK's significance stems from its crucial role in clathrin-mediated endocytosis, intracellular trafficking, and the dynamics of clathrin assembly/disassembly . Recent research has also revealed its vital function in controlling lysosomal dynamics through maintenance of lysosomal homeostasis during autophagy, making it an important target for studying cellular processes related to trafficking and degradation pathways .

What types of GAK antibodies are available for research applications?

Research-grade GAK antibodies come in multiple formats optimized for various experimental applications:

• Antibody Classes: Both monoclonal and polyclonal GAK antibodies are available . Monoclonal antibodies (like the mouse monoclonal clones 9-13 and 9-10) offer high specificity and reproducibility, while polyclonal antibodies provide broader epitope recognition .

• Host Species: Primarily rabbit polyclonal and mouse monoclonal options are available .

• Application Suitability: Different antibodies are validated for specific techniques as shown in the table below:

This diversity allows researchers to select the most appropriate antibody based on their specific experimental needs .

How do GAK antibodies differ in terms of epitope recognition?

GAK antibodies target different regions of the GAK protein, which affects their specificity and application suitability. For example, antibody ab190231 targets a synthetic peptide within human GAK amino acids 100-150 , while ab186120 recognizes a synthetic peptide within the 650-750 amino acid region . Some antibodies like A25860 specifically target the Q129 region of GAK . This epitope diversity is critical for experimental design, as it determines:

• The protein domain being detected (kinase domain, PTEN-like domain, J-domain, etc.)

• Accessibility of the epitope in different experimental conditions (native vs. denatured)

• Potential cross-reactivity with similar proteins or isoforms

Researchers should select antibodies whose epitope recognition aligns with their specific experimental goals, such as detecting specific domains, phosphorylation states, or protein interactions .

How can I validate GAK antibody specificity in knockout/knockdown models?

Validating GAK antibody specificity using genetic models is essential for ensuring result accuracy. A systematic validation approach includes:

• CRISPR/Cas9 Knockout Validation: Generate GAK-knockout cell lines using CRISPR/Cas9 genome editing. The knockout can be confirmed by Sanger sequencing to verify expected mutations and frameshifting in exons, as demonstrated in recent research . The absence of GAK protein expression in these knockout lines should be confirmed by immunoblotting with anti-GAK antibody.

• siRNA/shRNA Knockdown: For a complementary approach, perform transient knockdown using siRNA or stable knockdown using shRNA, followed by antibody testing. Look for reduced signal intensity proportional to knockdown efficiency.

• Rescue Experiments: Reconstitute GAK expression in knockout cells using lentiviral expression vectors containing GAK cDNA (like pLentiN-GAK). The vector can include a FLAG tag for independent detection. Compare antibody reactivity in wild-type, knockout, and reconstituted cells .

• Western Blot Controls: When performing validation Western blots, include peptide competition assays where the immunizing peptide blocks specific binding. For example, results from lane 2 in the validation data for antibody ab190231 showed absence of band when the antibody was pre-incubated with the synthesized peptide .

This multi-faceted validation approach ensures that observed signals are truly GAK-specific and not due to cross-reactivity with other proteins .

What are the optimal conditions for using GAK antibodies in immunofluorescence studies of clathrin-mediated endocytosis?

For successful immunofluorescence studies of GAK in clathrin-mediated endocytosis:

• Antibody Selection: Use monoclonal antibodies validated for immunofluorescence (IF), such as antibody clones 9-13 (A415) or 9-10 (A416), which have demonstrated reactivity in human and rat samples .

• Fixation Protocol:

  • For membrane structures and clathrin-coated vesicles: 4% paraformaldehyde (10 minutes at room temperature)

  • For preserving cytoskeletal interactions: Add 0.1% glutaraldehyde

  • Avoid methanol fixation which can disrupt membrane structures

• Permeabilization: Use 0.1% Triton X-100 for 5 minutes, which provides sufficient permeabilization while preserving vesicular structures.

• Co-localization Studies: For analyzing GAK's role in clathrin dynamics:

  • Co-stain with markers for clathrin (clathrin heavy chain antibody)

  • Use markers for early endosomes (EEA1) and recycling endosomes (Rab11)

  • Consider Hsc70 co-staining to visualize the uncoating process

• Signal Amplification: For studying low-abundance GAK populations, implement tyramide signal amplification or fluorescently labeled secondary antibody systems.

• Image Acquisition: Use high-resolution confocal microscopy with appropriate z-stacking to capture the dynamic 3D distribution of clathrin-coated vesicles and GAK.

When analyzing results, focus on temporal changes in GAK localization during vesicle formation, uncoating, and recycling, as GAK's association with these structures is transient and cell-cycle dependent .

How does GAK function in autophagy regulation and lysosomal homeostasis?

Recent research has uncovered GAK's critical role in autophagy through control of lysosomal dynamics:

• Autophagic Flux Regulation: Targeted disruption of GAK causes stagnation of autophagic flux. In GAK-depleted cells, autophagosomes accumulate due to impaired fusion with lysosomes, indicating that GAK is essential for the completion of autophagy .

• Lysosomal Homeostasis: GAK maintains lysosomal homeostasis through:

  • Regulation of lysosomal acidification

  • Control of lysosomal enzyme trafficking and activation

  • Maintenance of proper lysosomal membrane dynamics

• Mechanistic Pathway: GAK likely influences autophagy through its role in clathrin-mediated trafficking, which affects:

  • Proper sorting of lysosomal hydrolases

  • Trafficking of membrane components needed for autophagosome-lysosome fusion

  • Regulation of mTOR signaling components that control autophagy initiation

• Experimental Evidence: Studies using GAK-knockout A549 cells showed:

  • Increased LC3-II levels indicating autophagosome accumulation

  • Reduced degradation of autophagy substrates

  • Impaired lysosomal function and acidification

Understanding GAK's role in autophagy provides insight into fundamental cellular quality control mechanisms and offers potential therapeutic targets for diseases characterized by autophagy dysregulation .

What are the optimal conditions for Western blot analysis using GAK antibodies?

Achieving clear and specific detection of GAK (~143 kDa) by Western blot requires careful optimization:

• Sample Preparation:

  • Lyse cells in RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if studying GAK phosphorylation status

  • Sonicate briefly to shear DNA and reduce sample viscosity

  • For membrane fractions enriched in GAK, consider using a fractionation protocol

• Protein Separation:

  • Use 6-8% SDS-PAGE gels to properly resolve the 143 kDa GAK protein

  • Load 20-50 μg of total protein per lane

  • Include molecular weight markers spanning 100-250 kDa range

• Transfer Conditions:

  • Use wet transfer for large proteins like GAK (overnight at 30V, 4°C)

  • Consider adding 0.05% SDS to transfer buffer to improve large protein transfer

  • Use PVDF membrane with 0.45 μm pore size

• Antibody Conditions:

  • Primary antibody dilution: 1:500 for most GAK antibodies (e.g., ab190231)

  • Incubation: Overnight at 4°C with gentle rocking

  • Secondary antibody: HRP-conjugated anti-rabbit or anti-mouse IgG at 1:5000

  • Include positive controls (cell lines known to express GAK, such as 293 cells)

  • Negative controls should include peptide competition or GAK-knockout samples

• Detection:

  • Use enhanced chemiluminescence with extended exposure times (2-5 minutes)

  • For quantitative analysis, consider fluorescent secondary antibodies

Following these conditions typically yields a clear band at the expected molecular weight of 143 kDa, as demonstrated in validation data for antibodies such as ab190231 .

How can I optimize immunohistochemistry protocols for GAK detection in tissue samples?

For effective GAK detection in tissue samples through immunohistochemistry:

• Tissue Preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Embed in paraffin and section at 4-5 μm thickness

  • For human tissues (like testis samples used in ab190231 validation), ensure proper ethical approval and handling

• Antigen Retrieval:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) is effective for most GAK epitopes

  • Pressure cooker treatment for 20 minutes typically provides sufficient unmasking

  • Allow slides to cool in buffer for 20 minutes before proceeding

• Blocking and Antibody Incubation:

  • Block with 5% normal serum from the species of the secondary antibody

  • Add 0.3% hydrogen peroxide to quench endogenous peroxidase activity

  • Primary antibody concentration: Use antibodies validated for IHC (A416, A39113, A25860, ab190231)

  • Recommended dilution: 20 μg/ml for ab190231 as demonstrated in human testis tissue

  • Incubate at 4°C overnight in a humidified chamber

• Detection System:

  • Use polymer-based detection systems rather than avidin-biotin complexes for reduced background

  • Counterstain with hematoxylin to visualize tissue architecture

  • For multiplexing with other markers, consider fluorescence-based detection

• Controls and Validation:

  • Include positive control tissues known to express GAK

  • Use isotype controls to assess non-specific binding

  • Perform peptide competition assays to confirm specificity

  • Compare staining patterns with available literature on GAK expression patterns

This protocol can be adapted for both chromogenic and fluorescent detection based on experimental needs and available equipment .

What are the considerations for selecting between monoclonal and polyclonal GAK antibodies?

The choice between monoclonal and polyclonal GAK antibodies depends on specific research needs:

Decision Framework:

• For mechanistic studies examining specific domains or interactions, choose monoclonal antibodies directed at relevant epitopes

• For detection purposes in Western blots or IHC, polyclonal antibodies often provide stronger signals

• For co-localization studies, monoclonal antibodies minimize cross-reactivity concerns

• For cross-species studies, verify the conservation of epitopes in target species

How should I interpret contradictory results from different GAK antibodies?

When faced with contradictory results from different GAK antibodies, implement a systematic troubleshooting approach:

• Epitope Mapping Analysis:

  • Compare the epitopes recognized by each antibody (e.g., ab190231 targets aa 100-150 while ab186120 targets aa 650-750)

  • Different domains may be accessible in different experimental conditions

  • Some epitopes may be masked by protein-protein interactions in specific cellular contexts

• Validation Strategy:

  • Confirm results with knockout/knockdown controls for each antibody

  • Use peptide competition assays to verify specificity

  • Consider orthogonal detection methods (mass spectrometry) to confirm protein identity

• Technical Considerations:

  • Different antibodies may require different fixation methods, blocking agents, or detection systems

  • Clones like 9-13 (A415) work well for IP while others like A99719 are optimized for ELISA

  • Standardize protocols using positive control samples known to express GAK

• Biological Explanations:

  • Consider potential GAK splice variants or post-translational modifications

  • Different cellular compartments may show different accessibility to antibodies

  • Expression levels may vary with cell cycle (GAK peaks at G1 phase)

• Resolution Strategy:

  • When possible, use multiple antibodies targeting different epitopes

  • Prioritize results from antibodies with the most thorough validation

  • Discuss discrepancies transparently in publications

By systematically addressing these points, researchers can determine whether contradictions stem from technical limitations or reflect genuine biological complexity in GAK expression or function .

What quantitative approaches can I use to analyze GAK expression in relation to autophagic flux?

To quantitatively assess the relationship between GAK expression and autophagic flux:

• Western Blot Quantification:

  • Measure GAK protein levels using validated antibodies like ab190231

  • Simultaneously assess autophagy markers (LC3-II/LC3-I ratio, p62/SQSTM1 levels)

  • Use autophagy flux assays with lysosomal inhibitors (bafilomycin A1, chloroquine)

  • Normalize protein levels to appropriate loading controls (β-actin, GAPDH)

• Immunofluorescence Colocalization Analysis:

  • Perform double or triple labeling with GAK antibodies and autophagy markers

  • Calculate Pearson's or Mander's coefficients to quantify colocalization

  • Use high-content imaging to analyze hundreds of cells for statistical power

  • Implement particle analysis to count autophagosomes and autolysosomes

• Live Cell Imaging Approaches:

  • Create GAK-GFP fusion constructs for dynamic trafficking studies

  • Monitor autophagic flux using tandem fluorescent-tagged LC3 (tfLC3)

  • Measure temporal relationships between GAK recruitment and autophagosome formation/maturation

• Comparative Analysis in GAK-KO Models:

  • Compare autophagic flux measures in wild-type versus GAK-knockout cells

  • Perform rescue experiments with GAK constructs to establish causality

  • Analyze key timepoints in the autophagy pathway to pinpoint where GAK functions

• Mathematical Modeling:

  • Develop kinetic models of autophagic flux incorporating GAK activity

  • Use regression analysis to determine correlation between GAK levels and autophagic parameters

  • Apply machine learning approaches for complex pattern recognition

These methods, particularly when combined, provide robust quantitative data on how GAK influences autophagic flux, as supported by recent research showing that GAK disruption stagnates autophagic flux by disturbing lysosomal homeostasis .

How can computational modeling enhance our understanding of GAK antibody binding mechanisms?

Computational modeling offers powerful insights into GAK antibody binding mechanisms:

• Antibody Structure Prediction:

  • Leverage the relatively conserved structure of antibody domains to predict 3D structures

  • Implement homology modeling using tools like PIGS server or the knowledge-based AbPredict algorithm

  • Generate multiple models and refine through molecular dynamics simulations

• Epitope Mapping and Docking:

  • Use automated ligand docking to predict antibody-epitope interactions

  • Incorporate GAK's unique conformational preferences in docking protocols

  • Generate thousands of plausible options for antibody-antigen complexes

• Model Validation Through Experimental Data:

  • Validate computational models using site-directed mutagenesis of key residues

  • Apply saturation transfer difference NMR (STD-NMR) to define glycan-antigen contact surfaces

  • Use experimental data as metrics for selecting optimal 3D models from computationally generated options

• Application to GAK Research:

  • Model interactions between GAK domains (kinase, PTEN-like, J-domain) and specific antibodies

  • Predict potential cross-reactivity with related proteins

  • Design improved antibodies with enhanced specificity and affinity

• Combined Computational-Experimental Approach:

  • High-throughput techniques for characterizing structure and specificity

  • Define antibody binding site through quantitative assays and mutagenesis

  • Use computational screening against human glycome to validate specificity

This integrated approach has been successfully applied to characterize antibody-glycan complexes, providing a roadmap for improving understanding of GAK antibodies. The methodology combines experimental data with computational modeling to select the most likely binding models from thousands of possibilities .

What are common pitfalls in GAK antibody-based experiments and how can they be addressed?

Common challenges in GAK antibody experiments and their solutions include:

• High Molecular Weight Detection Issues:

  • Problem: Weak or absent signal for the 143 kDa GAK protein

  • Solution: Optimize transfer conditions for large proteins (longer transfer times, add 0.05% SDS to transfer buffer, use PVDF membrane with 0.45 μm pore size)

• Background or Non-specific Binding:

  • Problem: Multiple bands or high background in Western blots

  • Solution: Increase blocking time/concentration, optimize antibody dilution (typically 1:500 for most GAK antibodies like ab190231), include 0.1% Tween-20 in wash buffers

• Inconsistent Results Between Experiments:

  • Problem: Variable detection of GAK in replicate experiments

  • Solution: Standardize lysate preparation, include positive controls (293 cell lysate has been validated), and maintain consistent antibody lots

• Cross-reactivity Concerns:

  • Problem: Potential detection of proteins similar to GAK

  • Solution: Validate with GAK-knockout controls, perform peptide competition assays, verify bands appear at the expected 143 kDa size

• Epitope Accessibility in Fixed Tissues:

  • Problem: Poor signal in immunohistochemistry despite positive Western blot results

  • Solution: Optimize antigen retrieval (citrate buffer, pH 6.0), adjust antibody concentration (20 μg/ml for ab190231 has been validated), and extend incubation times

• Cell Cycle Variability:

  • Problem: Inconsistent GAK levels between samples

  • Solution: Synchronize cells when possible, as GAK expression peaks at G1 phase, or analyze expression relative to cell cycle markers

By anticipating these common issues and implementing appropriate controls and optimizations, researchers can significantly improve the reliability of GAK antibody experiments .

How can I design appropriate controls for GAK antibody experiments?

A comprehensive control strategy for GAK antibody experiments should include:

• Positive Controls:

  • Cell lines with verified GAK expression (293 cells have been validated)

  • Tissues known to express GAK (testis tissue has been validated for IHC)

  • Recombinant GAK protein or overexpression systems

• Negative Controls:

  • GAK-knockout cell lines generated via CRISPR/Cas9

  • siRNA/shRNA knockdown samples

  • Secondary antibody-only controls to assess non-specific binding

  • Isotype controls matching the primary antibody species and class

• Specificity Controls:

  • Peptide competition assays using the immunizing peptide

  • Comparison of multiple antibodies targeting different GAK epitopes

  • Western blots with predicted band size verification (143 kDa for full-length GAK)

• Technical Controls:

  • Loading controls appropriate for the application (β-actin, GAPDH for WB)

  • Tissue architecture markers for IHC (nuclear stains, tissue-specific markers)

  • Subcellular marker proteins for localization studies (clathrin, endosomal markers)

• Validation Through Multiple Techniques:

  • Confirm key findings with orthogonal methods (WB, IHC, IF)

  • Use genetic approaches (knockout/knockdown) to validate antibody specificity

  • Consider mass spectrometry validation for critical experiments

Implementation of this control strategy ensures that experimental results are robust, reproducible, and accurately reflect GAK biology rather than technical artifacts .

What quality control parameters should be assessed when validating a new GAK antibody lot?

When validating a new GAK antibody lot, assess these critical quality control parameters:

• Binding Specificity:

  • Western blot analysis against positive control lysates (e.g., 293 cells)

  • Verification of the expected 143 kDa band with minimal non-specific bands

  • Peptide competition assay showing elimination of specific signal

  • Testing against GAK-knockout controls where available

• Sensitivity and Signal-to-Noise Ratio:

  • Titration experiment with serial dilutions of antibody

  • Determination of optimal working concentration

  • Comparison of detection limits with previous lots

  • Assessment of background levels in negative control samples

• Reproducibility:

  • Technical replicates to assess consistency

  • Comparison with historical data from previous lots

  • Evaluation of batch-to-batch variation in staining patterns

• Application-Specific Performance:

  • For Western blot: band intensity, specificity, and molecular weight accuracy

  • For IHC: signal localization, background, and staining pattern in validated tissues (e.g., testis)

  • For IP: efficiency of target protein pulldown

  • For IF: subcellular localization pattern and colocalization with known markers

• Cross-Reactivity Assessment:

  • Testing across multiple relevant species (human, mouse, rat) if claimed

  • Evaluation in tissues/cells with varying GAK expression levels

  • Assessment of potential cross-reactivity with related proteins

• Documentation:

  • Certificate of analysis verification

  • Lot-specific validation data review

  • Documentation of any deviations from expected performance

This systematic validation approach ensures experimental reliability and facilitates troubleshooting if unexpected results occur. Always maintain records of antibody performance by lot number to track any changes over time .