CRK34 Antibody

What is the Crk protein and what role does it play in cellular signaling?

Crk belongs to a family of adaptor molecules widely expressed across numerous tissues. Despite lacking enzymatic activity, this protein plays a critical role in multiple signal transduction pathways leading to phosphorylation of various proteins. Crk forms multi-protein complexes via its SH2 (amino acids 13-118) and SH3 (amino acids 132-192 and 237-296) domains, which activate downstream molecules involved in cellular growth, differentiation, adhesion, and migration. Alternative splicing can generate additional isoforms, such as one lacking amino acids 205-304 .

For researchers investigating signaling pathways, it's important to understand that Crk participates in complex formation with various proteins, making it a crucial component to consider when studying cellular communication mechanisms. Experimental approaches should incorporate controls that account for these interactions to properly interpret results.

How does CD34 interact with adapter proteins like CrkL?

CD34 is a cell-surface transmembrane protein expressed specifically at the stem/progenitor stage of lymphohematopoietic development that appears to regulate adhesion. Research has demonstrated that CD34 couples specifically with the hematopoietic adapter protein CrkL. This interaction occurs at a membrane-proximal region of the CD34 tail, with CD34 binding specifically to the C-terminal SH3 domain of CrkL .

Interestingly, there appears to be differential specificity between CrkL and CrkII for CD34. While CD34 binds to CrkL, GST-CD34i full did not precipitate CrkII, despite CrkII being a highly homologous Crk family member. Furthermore, CD34i full does not bind other proteins known to associate with CrkL, such as c-Abl, c-Cbl, C3G, or paxillin, suggesting that CD34 directly interacts with the CrkL protein rather than participating in larger protein complexes .

What are the recommended storage conditions for Crk antibodies?

For optimal maintenance of antibody activity, use a manual defrost freezer and avoid repeated freeze-thaw cycles, as this can significantly degrade antibody performance. The following storage guidelines are recommended for Crk antibodies:

• 12 months from date of receipt, -20 to -70°C as supplied

• 1 month, 2 to 8°C under sterile conditions after reconstitution

• 6 months, -20 to -70°C under sterile conditions after reconstitution

These storage recommendations help maintain antibody integrity and ensure consistent experimental results across multiple investigations.

How can I validate the specificity of Crk antibodies in my experimental system?

Antibody validation is critical for reliable research outcomes. For Crk antibodies, Western blot analysis provides a fundamental validation approach. In published studies, specific bands for Crk were detected at approximately 46 kDa in multiple cell lines including HeLa (human cervical epithelial carcinoma), K562 (human chronic myelogenous leukemia), NIH-3T3 (mouse embryonic fibroblast), and MOLT-4 (human acute lymphoblastic leukemia) .

To properly validate a Crk antibody in your system:

• Include positive controls from well-characterized cell lines known to express Crk

• Use reducing conditions for SDS-PAGE (as demonstrated in referenced protocols)

• Compare results across multiple antibody concentrations (e.g., starting with 1 μg/mL)

• Confirm specificity using knockout/knockdown controls when possible

• Assess cross-reactivity with related proteins (especially CrkL and CrkII)

Remember that batch-to-batch variability can occur, as highlighted by Dr. David Rimm's experience where new batches of antibodies failed to reproduce original results despite being sold as identical . This emphasizes the importance of testing each new antibody lot against previously validated standards.

What techniques can be used to study the CrkL-CD34 interaction in primary cells?

Investigating the CrkL-CD34 interaction in primary cells requires specialized approaches:

• Co-immunoprecipitation: This technique verifies in vivo interactions. Use antibodies suitable for immunoprecipitation, as some antibodies (like 9C5) may be non-immunoprecipitating .

• GST-fusion protein precipitation: Construct GST-fusion proteins of the intracellular domain of CD34 (GST-CD34i) to precipitate interacting proteins from cell lysates. This approach successfully identified CrkL as a 39-kDa protein associating with CD34 .

• Domain mapping: To identify interaction domains, generate truncated constructs like GST-CrkL3′ (C-terminal SH3) and GST-CrkL5′ (N-terminal SH2SH3). This approach revealed that CD34 specifically binds the C-terminal SH3 domain of CrkL .

• Adhesion assays with antibody engagement: Treat cells with specific antibodies (15 μg/mL) and monitor changes in protein interactions during adhesion events. For inhibition studies, pre-treat with peptides (e.g., 250 μg/mL 9C5 peptide) 30 minutes prior to antibody treatment .

• Metabolic labeling: Use 35S-methionine/cysteine labeling to track newly synthesized proteins involved in the interaction complex .

How do phosphorylation states affect Crk/CrkL function and antibody detection?

Phosphorylation significantly impacts Crk/CrkL function and can affect antibody detection. For high-resolution separation of phosphorylated and phospho-deficient Crk/CrkL, incorporate Phos-tag acrylamide (such as FUJIFILM Wako Chemicals, NC0232095) into SDS-PAGE gels .

Key phosphorylation sites include Tyr221 on CrkII and Tyr207 on CrkL, which can be detected using site-specific antibodies (such as Cell Signaling #3491 for pCrkII-Tyr221 and Cell Signaling #34940 for pCrkL-Tyr207) . These phosphorylation events regulate protein interactions and downstream signaling.

When investigating phosphorylation-dependent interactions:

• Consider using pervanadate stimulation (2 minutes) to preserve phosphorylation states

• Use cold PBS and ice-cold lysis buffers with protease inhibitors to prevent dephosphorylation

• Compare results with general phosphotyrosine antibodies (like Cell Signaling #9411)

• Include both phosphorylated and non-phosphorylated controls in your experiments

What are common causes of inconsistent results when using Crk/CD34 antibodies?

Several factors can contribute to inconsistent results when working with Crk/CD34 antibodies:

• Batch-to-batch variability: As demonstrated by Dr. Rimm's experience, even antibodies sold by the same companies as supposedly identical products can yield dramatically different staining patterns . This variability represents one of the most significant challenges in antibody-based research.

• Cross-reactivity: Antibodies often recognize additional proteins beyond their intended targets. This is particularly relevant for Crk family proteins, which share significant homology. Always validate specificity for your specific application .

• Application-specific optimization: Optimal antibody dilutions vary between applications. As noted in protocols, "Optimal dilutions should be determined by each laboratory for each application" . Failure to optimize can lead to inconsistent results.

• Sample preparation variations: Differences in cell lysis procedures, buffer compositions, and protein denaturation can significantly impact antibody performance. Standardize these protocols across experiments.

• Detection system sensitivity: Different secondary antibodies and detection methods vary in sensitivity and can influence results. For example, some applications may require signal amplification using systems like the Tyramide Signal Amplification Kit .

How can researchers address batch-to-batch antibody variability in long-term studies?

To mitigate the impact of batch-to-batch antibody variability in longitudinal studies:

• Stockpile single batches: When initiating long-term studies, purchase sufficient antibody from a single batch to complete the entire project when possible.

• Perform comparative validation: Upon receiving new antibody batches, conduct side-by-side comparisons with previously validated batches using identical samples and protocols.

• Create internal reference standards: Develop well-characterized positive control samples that can be used to calibrate new antibody batches.

• Consider recombinant antibodies: As proposed by Bradbury and colleagues, recombinant antibodies defined to the DNA sequence level and manufactured in engineered cells may offer greater consistency, though at significantly higher cost .

• Document lot numbers and validation data: Maintain detailed records of antibody lot numbers, validation experiments, and performance metrics to track variability over time.

• Implement multiple detection approaches: Use complementary techniques (e.g., mass spectrometry) to verify key findings obtained with antibody-based methods.

What controls should be included when studying Crk/CrkL-CD34 interactions?

When investigating Crk/CrkL-CD34 interactions, include these essential controls:

• Negative controls for co-immunoprecipitation:

  • IgG isotype controls matched to the primary antibody species

  • Lysates from cells not expressing one of the interaction partners

  • Competitive inhibition with relevant peptides (e.g., CD34 peptide inhibition assays)

• Domain specificity controls:

  • GST-only precipitation to control for non-specific binding

  • Precipitation with truncated constructs (e.g., GST-CrkL3′ vs. GST-CrkL5′)

• Cross-interaction controls:

  • Test for interactions with related family members (e.g., CrkII when studying CrkL)

  • Verify absence of indirect interactions through common binding partners (c-Abl, c-Cbl, C3G, paxillin)

• Functional validation:

  • Adhesion assays with and without antibody engagement (15 μg/mL antibody)

  • Peptide competition experiments (pre-treat with 250 μg/mL peptide)

• Cell type controls:

  • Compare results across multiple cell lines (e.g., KG1a, HeLa, K562, NIH-3T3, MOLT-4)

How can researchers distinguish between direct and indirect protein interactions in Crk signaling complexes?

Distinguishing direct from indirect interactions in Crk signaling complexes requires systematic approaches:

• Domain mapping: Generate truncated constructs of both interaction partners to identify specific domains required for binding. The finding that CD34 binds specifically to the C-terminal SH3 domain of CrkL exemplifies this approach .

• In vitro binding assays: Perform direct binding assays with purified recombinant proteins. This eliminates cellular cofactors that might mediate indirect interactions.

• Competition assays: Use peptides corresponding to putative binding sites to competitively inhibit interactions. CD34 peptide inhibition assays have demonstrated that CrkL interacts at a membrane-proximal region of the CD34 tail .

• Cross-linking studies: Apply protein cross-linking followed by mass spectrometry to identify directly interacting protein regions at the amino acid level.

• Comparative binding studies: Evaluate binding to related proteins with high sequence homology. The differential specificity between CrkL and CrkII for CD34 suggests direct rather than indirect interaction (as indirect interactions would likely be preserved across highly homologous family members) .

• Control for known interaction partners: Test whether established binding partners of your protein of interest (e.g., c-Abl, c-Cbl, C3G, or paxillin for CrkL) co-precipitate in your experimental system .

What statistical approaches are recommended for quantifying Crk/CD34 expression in tissue samples?

For rigorous quantification of Crk/CD34 expression in tissue samples:

• Proliferation and apoptosis assessment: Calculate the ratios of marker-positive cells (e.g., Ki67, pHH3, TUNEL) to DAPI-positive cells to normalize for cell density variations .

• Morphometric analysis: Use ImageJ software (NIH) for standardized measurement of structural features, such as tissue dimensions and contact areas .

• Statistical testing:

  • Apply t-tests for pairwise comparisons between experimental groups

  • Use one-way ANOVA for comparisons involving three or more experimental groups

  • Consider non-parametric alternatives when data does not meet normality assumptions

• Blinded analysis: Implement blinded quantification where the scorer is unaware of sample identities to prevent unconscious bias.

• Replication strategy: Ensure biological replicates (different specimens) rather than just technical replicates (multiple measurements of the same specimen).

• Sample size determination: Conduct power analysis prior to experimentation to determine appropriate sample sizes for detecting biologically meaningful differences.

• Multiple hypothesis correction: Apply appropriate statistical corrections (e.g., Bonferroni, Benjamini-Hochberg) when testing multiple hypotheses to control false discovery rates.

How should researchers address contradictory findings between different antibody-based detection methods?

When faced with contradictory results between different antibody-based methods:

• Verify antibody specificity: Confirm that each antibody recognizes the intended target specifically in each application. Western blotting can verify that antibodies detect proteins of the expected molecular weight (e.g., 46 kDa for Crk) .

• Consider epitope accessibility: Different methods (Western blot, immunoprecipitation, immunohistochemistry) expose different protein epitopes. Some antibodies work well in denatured conditions but fail with native proteins, or vice versa.

• Evaluate fixation and sample preparation effects: Compare results using different fixation protocols and sample preparation methods, as these can dramatically affect epitope recognition.

• Implement orthogonal approaches: Utilize non-antibody-based methods (e.g., mass spectrometry, RNA-seq) to resolve contradictions through independent measurement technologies.

• Leverage genetic approaches: Use CRISPR/Cas9 knockout/knockin models to generate definitive controls for antibody specificity.

• Consider context-dependent protein modifications: Phosphorylation, glycosylation, or other post-translational modifications may affect antibody recognition in context-dependent ways. Use Phos-tag acrylamide in SDS-PAGE to separate phosphorylated and non-phosphorylated forms .

• Report contradictions transparently: When publishing, clearly document contradictory findings rather than selectively reporting results that align with a preferred hypothesis.