CRK35 Antibody

What are CRK and CRKL proteins, and why are they important targets for antibody-based research?

CRK and CRKL are adapter proteins involved in signal transduction pathways critical to cell proliferation, adhesion, and migration. CRK (v-crk avian sarcoma virus CT10 oncogene homolog) exists in multiple isoforms including Crk-I and Crk-II with a predicted molecular weight of approximately 42kDa. CRKL (v-crk sarcoma virus CT10 oncogene homolog-like) encodes a 303-amino acid polypeptide with a predicted molecular mass of 36 kDa . Both proteins contain SH2 and SH3 domains that mediate protein-protein interactions in signaling cascades. They are important research targets because they function as molecular switches in pathways related to cancer progression, immune response, and development. CRKL becomes phosphorylated when overexpressed, activates Ras-dependent and JNK pathways, and can transform fibroblasts . Understanding these proteins through antibody-based detection helps elucidate their roles in normal physiology and disease states.

What are the primary experimental applications for CRK/CRKL antibodies?

CRK/CRKL antibodies can be utilized across multiple experimental platforms:

For optimal results, researchers should select antibodies validated for their specific application. For example, the anti-CrkL antibody (PA1808) has been tested and validated for Flow Cytometry and Western Blot applications with demonstrated reactivity to human, mouse, and rat samples .

How should I determine which isoform-specific antibody to use for my CRK studies?

When selecting isoform-specific antibodies, consider:

• Research question specificity: Determine whether you need to distinguish between Crk-I, Crk-II, or Crk-III isoforms, which have different biological activities. For instance, Crk-II has less transforming activity than Crk-I, and mediates attachment-induced MAPK8 activation and cell motility in a Rac-dependent manner .

• Epitope location: Select antibodies raised against epitopes that can differentiate between isoforms. Antibodies targeting regions unique to specific splice variants will provide isoform selectivity.

• Validation evidence: Review immunogen information and published validation data. For example, MA5-15891 targets CRK using a purified recombinant fragment of human CRK expressed in E. coli .

• Cross-reactivity: Confirm the antibody has been tested for specificity against other related proteins, particularly CRKL, which shares structural homology with CRK proteins.

Always perform your own validation experiments to confirm isoform specificity in your experimental system, including Western blotting with positive and negative controls.

What are the optimal conditions for Western blotting with CRK/CRKL antibodies?

For successful Western blotting with CRK/CRKL antibodies:

• Sample preparation: Lyse cells in buffer containing appropriate phosphatase inhibitors, particularly when studying phosphorylated forms. CRKL is a substrate for BCR-ABL tyrosine kinase, making phosphorylation state preservation crucial for signaling studies .

• Gel separation: Use 10-12% polyacrylamide gels for optimal resolution of CRK/CRKL proteins (33-42 kDa).

• Transfer conditions: Semi-dry transfer (15-25V for 30-45 minutes) or wet transfer (100V for 1 hour) to PVDF membranes typically yields good results.

• Blocking: 5% non-fat dry milk in TBST works well, but for phospho-specific detection, switch to 5% BSA.

• Antibody dilutions: Start with manufacturer recommendations (typically 1:1000 for primary antibodies), then optimize. For anti-CrkL antibody PA1808, reconstitute with 0.2ml distilled water to yield a concentration of 500μg/ml, then dilute appropriately .

• Detection: Both chemiluminescence and fluorescence-based detection systems work well, with the latter offering better quantification capabilities.

If you encounter weak signals, consider increasing antibody concentration, extending incubation times, or enhancing the signal using more sensitive detection reagents.

How can I optimize immunofluorescence protocols for CRK/CRKL antibodies?

To achieve optimal immunofluorescence results with CRK/CRKL antibodies:

• Fixation: Test both paraformaldehyde (4%, 10-15 minutes) and methanol (-20°C, 10 minutes) fixation, as different epitopes may be preserved differently. For flow cytometry applications using anti-CRKL antibody, 4% paraformaldehyde fixation followed by permeabilization has proven effective .

• Permeabilization: Use 0.1-0.2% Triton X-100 for 5-10 minutes at room temperature for adequate access to intracellular epitopes.

• Blocking: 5-10% normal serum from the same species as the secondary antibody for 30-60 minutes helps reduce background. As demonstrated in flow cytometry protocols with PA1808, 10% normal goat serum effectively blocks non-specific binding .

• Primary antibody: Incubate at 4°C overnight or 1-2 hours at room temperature. Determine optimal concentration through titration (typically 1-5 μg/ml).

• Washing: Perform at least 3 washes of 5 minutes each with PBS containing 0.1% Tween-20 between all steps.

• Secondary antibody: Choose fluorophores compatible with your microscopy setup. For example, DyLight®488 conjugated secondary antibodies have been successfully used with CRKL antibodies .

• Nuclear counterstain: Include DAPI or Hoechst dye to visualize nuclei and provide context for CRK/CRKL localization.

• Mounting: Use anti-fade mounting medium to prevent photobleaching during imaging.

For multi-color imaging, carefully select fluorophores to minimize spectral overlap and include appropriate controls to assess bleed-through.

What are common troubleshooting strategies for inconsistent results with CRK/CRKL antibodies?

When encountering inconsistent results:

• Antibody validation: Confirm antibody specificity using positive and negative controls. Consider that the immunogen for anti-CRKL antibody PA1808 is a synthetic peptide at the C-terminus of human CRKL, which differs from mouse and rat sequences by one amino acid .

• Sample integrity: Ensure proper sample collection, storage, and preparation to preserve protein epitopes. For example, anti-CRKL antibody should be stored at -20°C for one year from receipt, and after reconstitution at 4°C for one month or aliquoted and stored at -20°C for six months, avoiding repeated freeze-thaw cycles .

• Protocol standardization:

  • Use consistent reagent lots

  • Standardize incubation times and temperatures

  • Prepare fresh working solutions

• Cross-reactivity assessment: Test for potential cross-reactivity with related proteins. Although anti-CRKL antibody PA1808 is listed as having no cross-reactivity with other proteins, this should be verified in your experimental system .

• Signal-to-noise optimization:

  • Adjust antibody concentrations

  • Modify blocking conditions

  • Increase wash stringency

• Batch effects minimization:

  • Process all experimental conditions simultaneously

  • Include internal standards across experiments

  • Normalize to housekeeping controls

• Documentation: Maintain detailed records of lot numbers, protocols, and any modifications to facilitate troubleshooting and reproducibility.

How can I use CRK/CRKL antibodies to study protein-protein interactions in signaling complexes?

To investigate CRK/CRKL protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use antibodies against CRK/CRKL to pull down protein complexes

  • Perform gentle lysis to preserve native interactions

  • Analyze precipitated complexes by Western blotting with antibodies against suspected interaction partners

• Proximity Ligation Assay (PLA):

  • Utilize paired antibodies against CRK/CRKL and potential interaction partners

  • This technique visualizes proteins within 40nm proximity

  • Quantify interaction signals at subcellular resolution

• FRET (Förster Resonance Energy Transfer):

  • Label CRK/CRKL and partner proteins with compatible fluorophores

  • Measure energy transfer as indication of physical proximity

  • Provides dynamic interaction information in living cells

• Immunofluorescence co-localization:

  • Perform double labeling with CRK/CRKL antibodies and antibodies against potential partners

  • Quantify co-localization using appropriate image analysis software

  • Consider that CRK-II is involved in phagocytosis of apoptotic cells and cell motility via interaction with DOCK1 and DOCK4

• Pull-down assays:

  • Use recombinant SH2 or SH3 domains from CRK/CRKL as baits

  • Identify binding partners through mass spectrometry

  • Confirm interactions using antibodies against specific candidates

For studying phosphorylation-dependent interactions, remember that CRKL is a substrate for the BCR-ABL tyrosine kinase , so phospho-specific antibodies may be necessary to capture transient, modification-dependent complexes.

What strategies can I use to study CRK/CRKL expression and function in tissue samples?

For tissue-based CRK/CRKL research:

• Immunohistochemistry optimization:

  • Test multiple fixation protocols (formalin, paraformaldehyde, alcohol-based)

  • Optimize antigen retrieval methods (citrate, EDTA, enzymatic)

  • Validate antibody specificity with appropriate tissue controls

  • Compare chromogenic vs. fluorescent detection systems

• Tissue microarray analysis:

  • Enables high-throughput screening across multiple tissue types

  • Standardizes staining conditions across samples

  • Facilitates quantitative comparison of expression levels

• Multiplex immunofluorescence:

  • Combine CRK/CRKL staining with cell type-specific markers

  • Include functional markers to correlate with signaling states

  • Use spectral unmixing for multiple fluorophores

• Laser capture microdissection:

  • Isolate specific CRK/CRKL-expressing cells or regions

  • Combine with protein extraction for Western blotting

  • Correlate with RNA analysis from parallel sections

• Spatial transcriptomics correlation:

  • Correlate CRK/CRKL protein expression with mRNA distribution

  • Map expression patterns to specific tissue microenvironments

  • Integrate with single-cell sequencing data

When analyzing results, consider that CRKL was identified as required for normal cellular responses to Fgf8, including survival and migration , which may influence interpretation of developmental or cancer tissue studies.

How can I use CRK/CRKL antibodies in flow cytometry for analyzing signaling dynamics?

For flow cytometry-based CRK/CRKL signaling analysis:

• Sample preparation optimization:

  • Fix cells with 4% paraformaldehyde and permeabilize with appropriate buffer

  • Block with 10% normal serum from the same species as secondary antibody

  • Incubate with anti-CRKL antibody (e.g., PA1808, 1 μg/1×10^6 cells) for 30 min at 20°C

• Phospho-flow cytometry:

  • Use phospho-specific antibodies to detect activated CRK/CRKL

  • Combine with total protein antibodies to normalize expression

  • Employ compatible fixation methods that preserve phospho-epitopes

• Time-course experiments:

  • Stimulate cells and fix at multiple timepoints

  • Track CRK/CRKL phosphorylation dynamics

  • Correlate with downstream pathway activation

• Multi-parameter analysis:

  • Combine CRK/CRKL staining with cell cycle markers

  • Add surface markers to identify specific cell populations

  • Include apoptosis indicators to correlate with cell survival

• Fluorescence compensation:

  • Carefully set up compensation controls for multicolor experiments

  • Use single-stained controls for each fluorophore

  • Consider spectral overlap when designing panels

• Quantification approaches:

  • Report median fluorescence intensity (MFI) rather than percent positive

  • Use standardized beads for day-to-day calibration

  • Apply appropriate statistical analysis for multiple samples

Remember that CRK-II mediates attachment-induced MAPK8 activation and cell motility , so integrating these pathways into your analysis can provide valuable functional context.

How do I interpret multiple bands observed in Western blots using CRK/CRKL antibodies?

When multiple bands appear in CRK/CRKL Western blots:

• Isoform identification:

  • CRK exists in multiple isoforms: Crk-I, Crk-II, and Crk-III with different molecular weights

  • Crk-I and Crk-II differ in their biological activities, with Crk-II having less transforming activity than Crk-I

  • The calculated molecular weight of CRKL is 33.8 kDa, but the observed molecular weight is typically around 39 kDa

• Post-translational modifications:

  • Phosphorylated forms may show mobility shifts

  • CRKL becomes phosphorylated when overexpressed and is a substrate for BCR-ABL tyrosine kinase

  • Other modifications like ubiquitination can produce higher molecular weight bands

• Proteolytic processing:

  • Sample preparation artifacts may cause protein degradation

  • Specific proteolytic events might generate functional fragments

• Non-specific binding:

  • Cross-reactivity with related proteins despite manufacturer claims

  • Secondary antibody binding to endogenous immunoglobulins

• Validation approaches:

  • Use lysates from knockdown/knockout cells as negative controls

  • Compare with recombinant protein standards of known molecular weight

  • Perform peptide competition assays with the immunizing peptide

  • Test multiple antibodies targeting different epitopes

For accurate interpretation, remember that the anti-CRKL antibody PA1808 immunogen is a synthetic peptide corresponding to a sequence at the C-terminus of human CRKL, which differs from mouse and rat sequences by one amino acid .

What is the significance of CRKL phosphorylation in disease-related research?

CRKL phosphorylation has important implications in multiple disease contexts:

• Cancer biology:

  • CRKL phosphorylation is a key event in BCR-ABL signaling in chronic myelogenous leukemia (CML)

  • CRKL was found to be a substrate for the BCR-ABL tyrosine kinase

  • Phosphorylated CRKL serves as a biomarker for BCR-ABL activity and treatment response

  • Quantifying phospho-CRKL by Western blotting or flow cytometry can monitor tyrosine kinase inhibitor efficacy

• Signal transduction research:

  • CRKL phosphorylation activates Ras-dependent and JNK pathways

  • This activation can lead to cellular transformation, making it relevant for oncogenesis studies

  • Temporal dynamics of phosphorylation correlate with specific cellular responses

• Developmental biology:

  • CRKL is required for normal cellular responses to Fgf8, including survival and migration

  • Phosphorylation status affects CRKL's ability to mediate these developmental signals

  • Embryological defects in CRKL-deficient models correlate with altered phosphorylation patterns

• Immunology applications:

  • CRKL phosphorylation influences immune cell migration and adhesion

  • Antibodies detecting phospho-CRKL can monitor immune activation states

  • Therapeutic interventions targeting this pathway can be assessed using phospho-specific antibodies

When designing experiments to study CRKL phosphorylation, consider using phosphatase inhibitors during sample preparation and phospho-specific antibodies for detection. Correlating total CRKL levels with phosphorylated forms provides insight into signaling efficiency and activation states.

How can I integrate CRK/CRKL antibody-based research with functional genomics approaches?

To comprehensively study CRK/CRKL biology by combining antibody-based detection with functional genomics:

• CRISPR/Cas9 knockout validation:

  • Generate CRK/CRKL knockout cell lines

  • Use antibodies to confirm protein depletion

  • Assess specificity by testing antibodies on knockout samples

  • Perform rescue experiments with wild-type and mutant constructs

• RNA interference correlation:

  • Knockdown CRK/CRKL using siRNA or shRNA

  • Correlate reduced protein levels (antibody detection) with mRNA depletion

  • Use antibodies to assess effects on downstream signaling proteins

• Overexpression systems:

  • Express tagged versions of CRK/CRKL proteins

  • Compare antibody detection of endogenous versus overexpressed proteins

  • Study phosphorylation states, as CRKL becomes phosphorylated when overexpressed

• Domain mutant analysis:

  • Generate SH2/SH3 domain mutants of CRK/CRKL

  • Use antibodies to assess effects on protein-protein interactions

  • Correlate with functional outcomes in pathways like Ras-dependent and JNK signaling

• Cellular phenotype correlation:

  • Quantify CRK/CRKL expression/phosphorylation by antibody-based methods

  • Correlate with phenotypes like cell motility, which is mediated by CRK-II in a Rac-dependent manner

  • Integrate with transcriptomic or proteomic profiles

• Systems biology integration:

  • Use antibody-validated interactions to build signaling network models

  • Correlate protein expression data with pathway activation signatures

  • Predict and test intervention points in CRK/CRKL-dependent pathways

This integrated approach provides mechanistic insights beyond what either antibody-based detection or genomic approaches could achieve alone.

How can I apply proximity-based labeling techniques using CRK/CRKL antibodies?

Proximity-based labeling offers powerful approaches for studying CRK/CRKL protein interactions:

• BioID and TurboID applications:

  • Generate CRK/CRKL fusion constructs with biotin ligase domains

  • Identify proximity partners through streptavidin pulldown and mass spectrometry

  • Validate interactions using CRK/CRKL antibodies

  • Particularly useful for identifying transient interactions in signaling complexes

• APEX2 proximity labeling:

  • Create CRK/CRKL-APEX2 fusion proteins

  • Perform peroxidase-catalyzed biotinylation of proximal proteins

  • Use antibodies against CRK/CRKL to confirm expression and localization of fusion proteins

  • Compare interactome data with known CRKL functions in Ras-dependent and JNK pathway activation

• Split-BioID strategies:

  • Fuse complementary BioID fragments to CRK/CRKL and suspected partners

  • Biotin labeling occurs only when proteins interact

  • Confirm protein expression and localization with specific antibodies

  • Especially valuable for studying dynamic assembly of signaling complexes

• Verification workflows:

  • Use conventional CRK/CRKL antibodies to validate proximity labeling results

  • Perform reciprocal experiments with different bait proteins

  • Correlate with co-immunoprecipitation data

  • Map interaction domains through mutational analysis

• Subcellular targeting:

  • Direct CRK/CRKL-BioID fusions to specific cellular compartments

  • Use antibodies to confirm correct localization

  • Compare interaction partners across different cellular locations

  • Particularly relevant as CRK-II mediates cell motility and membrane ruffling

These techniques can reveal novel insights into how CRK/CRKL functions within its signaling networks, particularly in contexts like apoptotic cell phagocytosis where CRK-II interacts with DOCK1 and DOCK4 .

What considerations are important when using CRK/CRKL antibodies in super-resolution microscopy?

When applying super-resolution microscopy to CRK/CRKL research:

• Antibody selection criteria:

  • Choose high-affinity, mono-specific antibodies

  • Select antibodies with minimal background staining

  • Consider directly conjugated primary antibodies to eliminate secondary antibody displacement error

  • Validate specificity using knockdown/knockout controls

• Technique-specific considerations:

  • For STORM/PALM: Select antibodies conjugated to photoswitchable fluorophores

  • For STED: Choose fluorophores with appropriate photostability

  • For SIM: Ensure high signal-to-noise ratio with minimal background

• Sample preparation optimization:

  • Test fixation methods beyond standard 4% paraformaldehyde

  • Consider specialized super-resolution fixatives (e.g., glutaraldehyde mixtures)

  • Optimize permeabilization to maintain structural integrity

  • Use appropriate blocking to minimize non-specific binding

• Multi-color imaging strategies:

  • Select fluorophores with minimal spectral overlap

  • Include proper controls for chromatic aberration

  • Consider sequential labeling approaches for crowded epitopes

  • Plan for registration correction between channels

• Quantification approaches:

  • Develop algorithms to quantify nanoscale clustering

  • Measure co-localization at super-resolution scale

  • Correlate distribution patterns with cellular structures

  • Consider 3D reconstruction for volumetric analysis

These approaches are particularly valuable for studying CRK/CRKL's role in membrane ruffling and cell motility mediated by CRK-II , where nanoscale organization at the membrane may be functionally significant.

What are the key considerations for ensuring reproducibility in CRK/CRKL antibody-based research?

To ensure reproducible CRK/CRKL antibody research:

• Antibody validation and documentation:

  • Confirm specificity using multiple methodologies

  • Document lot numbers and supplier information

  • Consider antibody validation initiatives guidelines

  • Share detailed methods including dilutions, incubation times, and buffer compositions

• Controls implementation:

  • Include positive and negative controls in every experiment

  • Use genetic knockdown/knockout samples when possible

  • Implement isotype controls for flow cytometry and immunohistochemistry

  • As shown in flow cytometry protocols, proper controls include isotype control antibody and unlabelled samples

• Protocol standardization:

  • Develop detailed, step-by-step protocols

  • Standardize sample collection and processing

  • Implement consistent quantification methods

  • Consider automated systems for reducing variability

• Reagent quality control:

  • Test new antibody lots against previous standards

  • Store antibodies according to manufacturer recommendations

  • For anti-CRKL antibody, store at -20°C for one year, and after reconstitution at 4°C for one month or aliquoted and frozen at -20°C for six months

  • Avoid repeated freeze-thaw cycles

• Data reporting standards:

  • Report all experimental conditions in publications

  • Include raw data when possible

  • Provide access to analysis scripts/methods

  • Follow field-specific reporting guidelines

By implementing these practices, researchers can improve the reliability and reproducibility of CRK/CRKL antibody-based research, contributing to a more robust understanding of these important signaling proteins in normal physiology and disease.

How might emerging technologies enhance CRK/CRKL antibody-based research in the future?

Several emerging technologies hold promise for advancing CRK/CRKL research:

• Single-cell proteomics integration:

  • Combine antibody-based detection with single-cell transcriptomics

  • Correlate CRK/CRKL protein levels with gene expression profiles

  • Study heterogeneity in signaling responses across cell populations

  • Particularly relevant for understanding CRKL's role in diverse cellular responses to Fgf8

• Spatial proteomics advancements:

  • Apply multiplexed antibody staining using cyclic immunofluorescence

  • Implement mass cytometry imaging for tissue analysis

  • Correlate CRK/CRKL distribution with tissue microenvironment

  • Map protein interactions in situ to understand contextual signaling

• Live-cell antibody applications:

  • Utilize cell-permeable nanobodies against CRK/CRKL

  • Track dynamic changes in localization and interaction

  • Monitor real-time phosphorylation using FRET-based biosensors

  • Directly observe CRK-II-mediated membrane ruffling and cell motility

• Antibody engineering innovations:

  • Develop recombinant antibodies with improved specificity

  • Create bispecific antibodies to study protein complexes

  • Engineer antibodies with reduced background for super-resolution imaging

  • Design conformation-specific antibodies to detect active signaling states

• Computational integration:

  • Develop machine learning algorithms for automated image analysis

  • Create predictive models of CRK/CRKL signaling networks

  • Integrate antibody-based data with multi-omics datasets

  • Apply systems biology approaches to understand network-level functions