CRK2 Antibody
Description
Definition and Overview of CRK2 Antibody
CRK2 antibodies are immunological tools designed to detect and study the CRKII protein (officially named CRK proto-oncogene, adaptor protein), a 34 kDa adaptor protein involved in intracellular signaling pathways. These antibodies enable researchers to investigate CRK2's role in regulating cellular processes such as cytoskeletal reorganization, immune cell activation, and stress responses . The monoclonal antibody A32498, for example, is a well-characterized mouse-derived reagent that binds specifically to human CRK2 in applications like Western blotting .
Applications in Biomedical Research
CRK2 antibodies facilitate critical investigations into:
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Immune Regulation: CRK2 modulates T-cell, B-cell, and natural killer (NK) cell functions by linking phosphorylated tyrosine residues to downstream effectors like p130Cas and Cbl .
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Cytoskeletal Dynamics: In Trypanosoma brucei, CRK2 phosphorylates β-tubulin to regulate microtubule assembly and cell morphology .
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Plant Stress Responses: Arabidopsis CRK2 interacts with aquaporins (e.g., PIP1-1, PIP2-2) and regulates callose deposition during salt stress .
Role in Podocyte Function
CRK2 forms hetero-oligomers with CRKL in kidney podocytes, as demonstrated by co-immunoprecipitation studies. Double knockout of CRK2/CRKL in mice caused proteinuria and impaired lamellipodia formation, highlighting their synergistic role in maintaining glomerular filtration .
Microtubule Regulation
CRK2 knockdown in Trypanosoma brucei led to cytoskeletal elongation (cell length increased from ~18.4 μm to ~30.2 μm) and microtubule branching, confirming its role in β-tubulin phosphorylation and cytoskeletal integrity .
Immune Signaling in Plants
CRK2 phosphorylates the NADPH oxidase RBOHD to regulate reactive oxygen species (ROS) production during pathogen defense. crk2 mutants showed 40–60% reduced ROS bursts and increased susceptibility to Pseudomonas syringae .
CRK2 Protein Interaction Network
Proteomic studies in Arabidopsis identified CRK2-binding partners involved in stress responses:
| Protein | Function | Interaction Frequency |
|---|---|---|
| PLDα1 | Phospholipid signaling | 8/8 replicates |
| Aquaporin PIP1-3 | Water transport | 8/8 replicates |
| ATPase 11 | Ion homeostasis | 8/8 replicates |
| CALS1/CALS3/CALS12 | Callose synthase activity | 1/8 replicates |
This network suggests CRK2’s role in coordinating membrane and osmotic stress responses .
Future Directions and Therapeutic Potential
CRK2 antibodies are pivotal for exploring:
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Cancer Immunotherapy: CRK2’s regulatory role in NK cell cytotoxicity positions it as a target for enhancing immune responses .
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Tubulin-Targeted Therapies: Inhibiting CRK2-mediated β-tubulin phosphorylation could disrupt pathogen cytoskeletons .
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Agricultural Biotechnology: Engineering CRK2 variants may improve crop resilience to salinity and pathogens .
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Inhibits the interaction between GARU and GID1A by phosphorylating GARU. GARU is an E3 ubiquitin ligase that mediates the degradation of GID1. PMID: 29042542
Q&A
Experimental Design for CRK2 Antibody Studies
Q: How should I design experiments to study the role of CRK2 using CRK2 antibodies in cell signaling pathways? A: When designing experiments to study CRK2, consider using CRK2 antibodies for immunoprecipitation or Western blotting to assess protein expression levels. Additionally, use siRNA or CRISPR-Cas9 to knockdown CRK2 and observe changes in signaling pathways. For more detailed analysis, employ co-immunoprecipitation to identify interacting proteins.
Data Interpretation and Contradiction Analysis
Q: How do I resolve discrepancies in data when using CRK2 antibodies across different experimental conditions? A: Discrepancies can arise from variations in antibody specificity, experimental conditions, or sample preparation. To resolve these, ensure consistent antibody batches are used, validate antibody specificity through controls, and standardize experimental protocols. Consider using orthogonal methods like mass spectrometry to confirm findings.
Advanced Research Questions: CRK2 Antibody Specificity
Q: What strategies can be employed to enhance the specificity of CRK2 antibodies for advanced research applications? A: To enhance specificity, consider using peptide competition assays to validate antibody binding. Additionally, epitope mapping can help identify unique binding sites on CRK2, reducing cross-reactivity. For advanced applications, antibody engineering techniques like chimerization or humanization can improve specificity and reduce immunogenicity.
Methodological Considerations for CRK2 Antibody Use
Q: What are the key methodological considerations when using CRK2 antibodies for immunohistochemistry (IHC) or immunofluorescence (IF)? A: For IHC and IF, optimize fixation and permeabilization conditions to preserve antigenicity. Use appropriate blocking agents to reduce background staining. Validate antibody specificity by using negative controls or competing peptides. Consider using secondary antibodies conjugated to fluorescent dyes or enzymes for enhanced detection.
Troubleshooting Common Issues with CRK2 Antibodies
Q: How can I troubleshoot common issues such as low signal or high background when using CRK2 antibodies? A: Low signal may be due to insufficient antigen retrieval or inadequate antibody concentration. High background can result from non-specific binding; try optimizing blocking conditions or using a different secondary antibody. Consider using alternative detection methods like chemiluminescence or fluorescence to enhance signal-to-noise ratio.
Advanced Techniques for CRK2 Antibody-Based Research
Q: What advanced techniques can be used in conjunction with CRK2 antibodies to study protein-protein interactions? A: Techniques like proximity ligation assay (PLA) or BioID can be used to study protein-protein interactions involving CRK2. PLA allows for the detection of protein complexes in situ, while BioID enables the identification of interacting proteins through proximity-dependent biotinylation.
Quantitative Analysis Using CRK2 Antibodies
Q: How can I perform quantitative analysis of CRK2 expression using CRK2 antibodies? A: For quantitative analysis, use techniques like quantitative Western blotting or flow cytometry. Ensure that standards or controls are included to normalize data. Consider using software tools to analyze band intensities or fluorescence signals.
Cross-Reactivity and Specificity Concerns
Q: How can I assess and mitigate potential cross-reactivity issues with CRK2 antibodies? A: Assess cross-reactivity by testing the antibody against closely related proteins or using peptide arrays. Mitigate cross-reactivity by optimizing antibody concentrations and using blocking agents. Consider using antibodies with well-characterized epitopes or employing orthogonal validation methods.
CRK2 Antibody Stability and Storage
Q: What are the best practices for storing and handling CRK2 antibodies to maintain their stability and efficacy? A: Store CRK2 antibodies at -20°C or -80°C to prevent degradation. Avoid repeated freeze-thaw cycles, and use sterile conditions to prevent contamination. Consider aliquoting antibodies to minimize exposure to air and moisture.
Future Directions in CRK2 Antibody Research
Q: What future directions or emerging technologies could enhance the utility of CRK2 antibodies in research? A: Emerging technologies like single-molecule localization microscopy (SMLM) or super-resolution microscopy can enhance spatial resolution when using CRK2 antibodies. Additionally, advances in antibody engineering and nanobody technology may provide more specific and versatile tools for studying CRK2.
Example Data Table: CRK2 Antibody Validation
| Antibody | Epitope | Specificity | Sensitivity |
|---|---|---|---|
| CRK2-1 | N-terminal | High | 1:1000 |
| CRK2-2 | C-terminal | Moderate | 1:500 |
Detailed Research Findings: CRK2 in Cell Signaling
CRK2 plays a crucial role in cell signaling pathways, particularly in the regulation of cytoskeletal dynamics and cell migration. Studies using CRK2 antibodies have shown that CRK2 interacts with various adaptor proteins to modulate signaling cascades. For instance, CRK2 can bind to DOCK180, enhancing Rac1 activation and promoting cell migration. Understanding these interactions is critical for elucidating the mechanisms underlying CRK2's role in cellular processes.
