CRK7 Antibody

What is the difference between CRK7 and Cytokeratin 7 antibodies?

There is often confusion between these two distinct antibodies in research literature. CRK7 (CDC2 related protein kinase 7/CDK12) antibodies target a kinase involved in cell signaling pathways , whereas Cytokeratin 7 (CK7) antibodies recognize an intermediate filament protein expressed in epithelial cells . These antibodies have different applications based on their target proteins, with CK7 antibodies being particularly valuable in diagnostic pathology and CRK antibodies being important in immune response research .

What are the structural characteristics of Cytokeratin 7 that make it a useful diagnostic marker?

Cytokeratin 7 is an intermediate filament protein of approximately 51-54 kDa that recognizes simple epithelium found in glandular and transitional epithelia, but not in stratified squamous epithelia . Its specific expression pattern in epithelial cells of ovary, lung, and breast makes it an excellent diagnostic marker . CK7 is particularly useful because it is expressed in the epithelia lining the cavities of internal organs, gland ducts, and blood vessels, creating a distinctive staining pattern that helps differentiate tissue origins in pathological examination .

How does the CRK family function in immune response regulation?

The CRK family proteins (CrkI, CrkII, and CrkL) are adaptor proteins that act as critical signal molecules in immune cell functions . Research indicates that CRK proteins can function as a two-way molecular switch controlling NK cell-mediated cytotoxicity . When phosphorylated at specific tyrosine residues (Tyr221 in CrkII, Tyr207 in CrkL), these proteins undergo conformational changes that regulate their interactions with other signaling molecules . During NK cell inhibition, phosphorylated Crk dissociates from scaffold proteins like p130Cas and Cbl protein, demonstrating their regulatory role in immune cell signaling .

What are the optimal protocols for using Cytokeratin 7 antibodies in immunohistochemistry?

For optimal CK7 antibody performance in immunohistochemistry, researchers should consider the following methodology:

• Antigen retrieval: TE buffer at pH 9.0 is suggested, though citrate buffer at pH 6.0 can also be effective .

• Dilution ratios: For IHC applications, a dilution range of 1:500-1:2000 is recommended for monoclonal CK7 antibodies .

• Tissue preparation: Formalin-fixed paraffin-embedded tissue sections provide reliable results for CK7 detection .

• Detection systems: Both conventional immunoperoxidase and fluorescence-based detection systems can be employed, with the rabbit monoclonal CK7 antibody (clone BC1) showing superior staining intensity compared to mouse monoclonal alternatives .

Researchers should validate these parameters for their specific tissue types and experimental conditions.

How can CRK7/CK7 antibodies be optimized for multiplex immunostaining applications?

Multiplex immunostaining with CK7 antibodies can be achieved through:

• Antibody cocktails: CK7 can be effectively combined with CDX-2 in an antibody cocktail for 4-step double stain assays .

• Compatible marker selection: CK7 functions well with CK20, CDX2, and TTF1 in multiplex panels for distinguishing primary adenocarcinomas from metastatic ones .

• Signal optimization: When using fluorescent conjugates like Alexa Fluor 488-conjugated anti-CK7 , researchers should implement cyclical bleaching protocols for imaging multiple markers across several cycles.

• Cross-reactivity prevention: Careful selection of antibodies raised in different host species (rabbit vs. mouse) can minimize cross-reactivity issues in multiplex applications .

What are the validated applications for recombinant antibodies against human tissue kallikreins related to CRK7?

Research on recombinant antibodies against human tissue kallikreins, particularly KLK7 (which should not be confused with CK7), has demonstrated several validated applications:

• Enzyme inhibition assays: Single-chain variable fragment antibodies (scFv) selected from human naïve phage display libraries can be characterized for their inhibitory activities against kallikreins .

• Affinity maturation: Low-IC50 scFv antibodies can be further affinity matured to generate more specific antibodies for target proteases .

• Expression systems: scFv-Fc format expression in HEK293-6E cells allows for effective production of recombinant antibodies .

• Therapeutic development: Phage-display methodology combined with enzymology assays provides a suitable approach for developing inhibitors for kallikreins as potential therapeutics for diseases related to uncontrolled kallikrein activity .

How should researchers design experiments to investigate Crk protein phosphorylation dynamics in NK cell signaling?

When investigating Crk protein phosphorylation dynamics in NK cell signaling, researchers should design experiments that:

• Monitor specific phosphorylation sites: Focus on Tyr221 in CrkII and Tyr207 in CrkL, which are critical for the negative regulation of signaling pathways .

• Implement FRET-based probes: Consider fluorescence resonance energy transfer approaches using truncated forms of CrkL sandwiched with fluorescent proteins (like Venus and ECFP) to detect conformational changes upon phosphorylation .

• Analyze temporal dynamics: Examine when and where phosphorylated Crk dissociates from signaling molecules at immunological synapses during inhibition processes .

• Include appropriate controls: Compare NK cells stimulated with 721.221 cells versus unstimulated controls to properly assess activation-dependent changes .

The table below summarizes key signal molecules associated with Crk in immune cells:

What considerations are important when validating the specificity of novel Cytokeratin 7 antibodies?

Validation of novel CK7 antibody specificity requires a comprehensive approach:

• Multi-platform validation: Assess antibody performance using ELISA against CK7 peptide, Western blotting on appropriate cell lysates (e.g., HeLa cells), and immunohistochemical staining on formalin-fixed paraffin-embedded tissue sections .

• Cross-reactivity testing: Verify that the antibody shows no cross-reaction with other intermediate filament proteins .

• Comparative analysis: Test against established antibodies (e.g., comparing novel rabbit monoclonal CK7 antibodies with the mouse monoclonal OV-TL 12/30) .

• Tissue panel screening: Evaluate staining patterns across a variety of human tissues known to either express or lack CK7, including lung, breast, ovarian, and colon tissues .

• Detection of expected molecular weight: Confirm identification of the correct 51-54 kDa protein via Western blot analysis .

How can researchers integrate CK7 expression data with other molecular markers for improved cancer subtyping?

For advanced cancer subtyping using CK7 along with other molecular markers:

• Design marker panels: Combine CK7 with complementary markers such as CK20, CDX-2, and TTF1 to create discriminatory panels .

• Implement scoring systems: Develop quantitative scoring methods that account for both staining intensity and percentage of positive cells.

• Correlate with genomic data: Integrate immunohistochemical findings with genomic profiling to identify relationships between protein expression and genetic alterations.

• Utilize digital pathology: Employ image analysis algorithms to quantitatively assess CK7 expression patterns across different tumor types.

The following expression pattern is commonly used in diagnostic pathology:

• Pulmonary, ovarian, and breast carcinomas: CK7+

• Most colon carcinomas: CK7-

What are common sources of false positive or negative results when using CK7 antibodies?

Understanding potential pitfalls in CK7 antibody applications:

• False positives:

  • Cross-reactivity with other cytokeratins in poorly fixed tissues

  • Non-specific binding due to inappropriate antibody concentrations (optimal dilution range for IHC: 1:500-1:2000)

  • Endogenous peroxidase activity if not properly blocked

• False negatives:

  • Inadequate antigen retrieval (requires TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Tissue fixation issues affecting epitope accessibility

  • Degradation of CK7 protein in poorly preserved specimens

  • Antibody storage conditions affecting stability (requires storage at -20°C)

How should researchers interpret conflicting CK7 expression data between different experimental platforms?

When facing discrepancies in CK7 expression results across different platforms:

• Consider technical variations: Different detection methods (IHC vs. IF vs. Western blot) have varying sensitivities and specificities .

• Evaluate antibody clone specificity: Different antibody clones (e.g., BC1 vs. OV-TL 12/30) may recognize different epitopes on the CK7 protein .

• Assess specimen preparation: Variations in fixation protocols can affect epitope preservation and accessibility.

• Quantification approaches: Differences in scoring methods between manual assessment and digital image analysis can lead to apparent discrepancies.

• Biological heterogeneity: Intratumoral heterogeneity may result in sampling variability between different specimens from the same tumor.

What experimental controls are essential when investigating the role of Crk phosphorylation in immune cell regulation?

Essential controls for Crk phosphorylation studies include:

• Phosphorylation-specific controls:

  • Phosphatase treatment controls to confirm specificity of phospho-specific antibodies

  • Site-directed mutagenesis of key tyrosine residues (Y221 in CrkII, Y207 in CrkL) to non-phosphorylatable phenylalanine

• Stimulation controls:

  • Positive controls using appropriate stimuli known to induce Crk phosphorylation

  • Time-course experiments to capture dynamic phosphorylation events

• Specificity controls:

  • siRNA or CRISPR-mediated knockdown/knockout of Crk to validate antibody specificity

  • Immunoprecipitation followed by mass spectrometry to confirm identified proteins

• Functional validation:

  • Correlate phosphorylation status with downstream functional outcomes (e.g., NK cell cytotoxicity, T cell activation)

What emerging technologies might enhance the utility of CK7/CRK antibodies in biomedical research?

Emerging technologies poised to transform CK7/CRK antibody applications include:

• Highly multiplexed imaging systems allowing simultaneous detection of dozens of markers in single tissue sections

• Single-cell proteomics approaches to correlate CK7 expression with comprehensive proteomic profiles

• CRISPR-based functional genomics to systematically investigate the role of CK7 and CRK family proteins in cellular processes

• Advanced recombinant antibody engineering techniques to develop more specific and sensitive detection reagents

• Cyclical immunofluorescence methodologies that enable imaging of multiple markers through repeated rounds of staining, imaging, and bleaching

How might the understanding of CRK signaling pathways in immune cells lead to novel therapeutic approaches?

The potential therapeutic implications of CRK signaling research include:

• Targeting Crk-mediated signaling pathways to modulate NK cell activity in cancer immunotherapy

• Developing inhibitors that selectively disrupt specific Crk-protein interactions while preserving others

• Utilizing the "molecular switch" property of Crk proteins to engineer controllable immune cell responses

• Exploring the relationship between Crk phosphorylation status and responses to existing immunotherapies to identify predictive biomarkers

As our understanding of these complex signaling networks grows, the potential for translating basic research on CRK family proteins into therapeutic applications continues to expand.