CPK7 Antibody

What is the molecular target of CPK7 antibody and what is its biological significance?

When referring to Cytokeratin 7 antibodies, the target is a type II intermediate filament protein with a molecular weight of approximately 51kDa. CK7 has significant biological functions including blocking interferon-dependent interphase and stimulating DNA synthesis in cells. It's also involved in the translational regulation of human papillomavirus type 16 E7 mRNA (HPV16 E7) .

For calcium-dependent protein kinase 7 antibodies, the target belongs to the CPK family, which contains EF-hand Ca²⁺-binding motifs and a Ser/Thr kinase domain that integrate both Ca²⁺ sensing and responding activity within a single protein. While the search results don't specifically describe CPK7, related CPKs like CPK28 play crucial roles in Ca²⁺-dependent signal transduction pathways .

What is the expression pattern of CPK7 in normal versus pathological tissues?

Cytokeratin 7 shows distinct expression patterns that are valuable for diagnostic applications. In colorectal carcinoma (CRC), tumors are typically CK7− and CK20+, but a subset of CRCs express CK7+ . This aberrant expression pattern has significant prognostic implications, as CK7+ CRC tumors are associated with shorter cancer-specific survival (restricted mean 4.98 vs. 7.74 years, P = 0.007) and reduced 5-year survival rates (29.4% vs. 64.6%, P = 0.0221) compared to CK7− tumors .

For calcium-dependent protein kinases, expression patterns vary by isoform and tissue type. Similar proteins like CPK28 exhibit rapid activation within seconds of environmental stimuli such as cold shock in plants .

How is CPK7 antibody specificity validated in research applications?

Specificity validation for antibodies targeting Cytokeratin 7 typically includes:

• Confirming recognition of the target protein at the expected molecular weight (~51kDa for CK7) via Western blotting

• Verifying target reactivity in appropriate cell/tissue types

• Employing multiple antibody clones (such as 5D12 for CK7) to confirm specificity

• Using techniques like ELISA and FACS to confirm binding characteristics

For novel antibodies, validation may include hybridoma development, isotope testing (e.g., IgG2a/κ), and affinity constant (Kaff) measurement via non-competitive ELISA, similar to protocols used for other critical cellular proteins .

What are the optimal sample preparation protocols for CPK7 antibody in different research applications?

For immunohistochemistry with CK7 antibodies:

• Formalin-fixed, paraffin-embedded (FFPE) tissue sections are standard for CK7 detection

• The tissue microarray method has been successfully employed for analyzing CK7 expression in large sample cohorts

• Positivity thresholds should be established (>10% positive tumor cells has been used as a cutoff for CK7 positivity in colorectal cancer studies)

For protein-protein interaction studies with calcium-dependent protein kinases:

• In vitro pull-down assays using GST-tagged kinase and His-tagged potential interactors

• Coimmunoprecipitation (co-IP) assays using epitope-tagged proteins (HA-FLAG, Myc)

• Bimolecular fluorescence complementation (BiFC) for visualizing interactions in vivo

How can researchers distinguish between true CPK7 signal and background in immunohistochemistry?

When evaluating CK7 immunostaining:

• Establish clear positivity thresholds based on literature and controlled experiments (>10% positivity for CK7 is commonly used)

• Include proper positive controls (known CK7-expressing tissues) and negative controls

• Compare staining patterns with expected subcellular localization

• Use parallel staining with complementary markers (like CK20 in colorectal tissues) for contextual evaluation

• Consider dual-staining approaches to confirm cell-type specific expression

What cross-reactivity concerns exist with CPK7 antibodies and how can they be mitigated?

For Cytokeratin 7 antibodies:

• Potential cross-reactivity with other cytokeratin family members due to structural similarities

• Mitigation strategies include using monoclonal antibodies with validated specificity like clone 5D12

• Sequence alignment analyses can help predict potential cross-reactivity

• Testing against panels of related proteins (other cytokeratins) is recommended

For calcium-dependent protein kinases:

• The CPK family has 34 members in Arabidopsis with overlapping and distinct expression patterns

• Use highly specific antibodies that target unique regions outside the conserved kinase domain

• Validate specificity using knockout/knockdown controls when possible

How should researchers interpret discrepancies between CPK7 protein expression and mRNA levels?

When facing discrepancies between protein and transcript levels:

• Consider post-transcriptional regulation mechanisms. For example, NLP7 (a transcription factor interacting with CPK28) shows minimal changes in transcript and protein levels during early cold exposure despite significant functional changes due to subcellular relocalization .

• Evaluate protein stability and turnover. Some proteins maintain stable levels despite transcriptional changes due to long half-lives or regulated degradation.

• Assess protein localization changes rather than total expression. As demonstrated with NLP7, functionality can change dramatically through subcellular redistribution without altering total protein levels .

• Use multiple detection methods. Combine Western blotting, immunohistochemistry, and mass spectrometry approaches to build a comprehensive understanding of expression patterns.

What statistical approaches are most appropriate for analyzing CPK7 expression data in prognostic studies?

Based on established research protocols for CK7 expression analysis:

• Kaplan-Meier survival analysis with log-rank tests for comparing survival outcomes between CK7+ and CK7− groups (as used in colorectal cancer studies)

• Multivariate Cox regression models to control for confounding variables such as:

  • Tumor stage and grade

  • Anatomical location (e.g., right vs. left-sided tumors)

  • Other molecular markers (e.g., MMR status)

• Restricted mean survival time analysis, which showed significant differences in CK7+ vs. CK7− tumors (4.98 vs. 7.74 years)

• Categorical analysis using established cutoff thresholds (>10% positivity for CK7, >25% for CK20)

How can researchers integrate CPK7 expression data with other molecular markers for comprehensive pathway analysis?

For integrated pathway analysis:

• Combine CK7 status with complementary markers like CK20 to establish comprehensive immunoprofiles. In colorectal cancer, the standard profile is CK7−/CK20+, with variations having prognostic implications .

• Correlate CPK7 expression with functional pathway indicators:

  • For calcium-dependent kinases, analyze relationships with downstream phosphorylation targets

  • For cytokeratins, examine correlations with epithelial-mesenchymal transition markers

• Apply multiparametric analysis incorporating:

  • Mismatch-repair (MMR) status, which shows significant prognostic value in CRC (MMR-deficient tumors exhibited longer survival)

  • Tumor laterality (right vs. left-sided), which correlates with both MMR status and CK expression patterns

  • Stage and grade information

• Use network analysis software to identify functional relationships between CPK7 and other molecular markers, particularly for calcium-dependent kinases that participate in complex signaling cascades .

How can phosphorylation state-specific CPK7 antibodies be developed and validated?

For developing phosphorylation state-specific antibodies for calcium-dependent protein kinases:

• Identify key phosphorylation sites through mass spectrometry analysis similar to the approach used for identifying CPK28 phosphorylation targets .

• Generate antibodies against synthetic phosphopeptides corresponding to specific phosphorylation sites.

• Validate specificity using:

  • Comparison of phosphorylated vs. non-phosphorylated recombinant proteins

  • Phosphatase-treated controls to confirm phospho-specificity

  • In vitro kinase assays to generate defined phosphorylation states

  • Knockout/knockdown controls to confirm antibody specificity

• Test antibody performance in detecting activation-dependent phosphorylation, such as the rapid activation of CPK28, which occurs within 10 seconds of cold shock in a Ca²⁺-dependent manner .

What are the methodological considerations for studying CPK7's role in protein-protein interaction networks?

Based on established protocols for related proteins:

• Initial identification of interacting partners:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using tagged CPK7 protein, similar to the approach used to identify CPK28-NLP7 interactions

  • Yeast two-hybrid screening for novel interactors

• Confirmation of direct interactions:

  • In vitro pull-down assays using purified recombinant proteins (GST-tagged or His-tagged)

  • Coimmunoprecipitation (co-IP) in relevant cell systems

• Visualization of interactions in cellular contexts:

  • Bimolecular fluorescence complementation (BiFC) to determine subcellular localization of interactions

  • Förster resonance energy transfer (FRET) to assess dynamic interactions in live cells

• Functional validation of interactions:

  • In vitro phosphorylation assays to identify substrates

  • Mutational analysis of key residues to disrupt specific interactions

What techniques can be employed to study the dynamic regulation of CPK7 in response to cellular signals?

For calcium-dependent protein kinases, these approaches are recommended:

• Real-time activation monitoring:

  • In-gel kinase assays using specific substrates to measure activity changes in response to stimuli (as demonstrated for CPK28 activation within 10 seconds of cold shock)

  • FRET-based biosensors to monitor conformational changes upon Ca²⁺ binding

• Subcellular localization dynamics:

  • Live-cell imaging with fluorescently tagged proteins

  • Cell fractionation assays to quantify nuclei-cytoplasmic partitioning in response to stimuli

  • Immunofluorescence with phospho-specific antibodies to track activated forms

• Signal-dependent interaction studies:

  • Proximity labeling approaches (BioID, APEX) to capture transient interactions

  • Quantitative proteomics with SILAC to measure interaction dynamics

  • Temporal analysis of protein complex formation using sequential co-IP assays

• Functional consequences of activation:

  • Phosphoproteomics to identify downstream substrates

  • Transcriptome analysis to assess impacts on gene expression

  • Correlation of kinase activity with physiological responses (such as cold tolerance)

What are common technical challenges when using CPK7 antibodies in Western blot applications and how can they be addressed?

Common challenges and solutions:

• Multiple bands or non-specific binding:

  • Optimize primary antibody concentration (typically starting with 1:1000 dilution for monoclonal antibodies)

  • Increase blocking stringency using 5% BSA or milk in TBS-T

  • Include competitive blocking with immunizing peptide to confirm specificity

  • Use gradient gels to better resolve proteins of similar molecular weight

• Weak or absent signal for Cytokeratin 7 (~51kDa):

  • Verify sample preparation methods preserve protein integrity

  • Optimize protein loading (20-50μg total protein typically)

  • Consider native vs. denaturing conditions depending on antibody epitope

  • Test multiple antibody clones with different epitope recognition

• High background:

  • Increase washing duration and frequency between antibody incubations

  • Validate secondary antibody compatibility and specificity

  • Consider using HRP-labeled MAbs for direct detection

How can researchers optimize immunohistochemical protocols for CPK7 detection in different tissue types?

Optimization strategies include:

• Antigen retrieval optimization:

  • Compare heat-induced epitope retrieval methods (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Adjust retrieval duration based on tissue fixation conditions

  • Consider proteolytic enzyme-based retrieval for heavily fixed samples

• Detection system selection:

  • For weakly expressed targets, employ polymer-based detection systems

  • For multiplex staining, use spectrally distinct fluorescent secondaries

  • Consider tyramide signal amplification for low abundance targets

• Validation across tissue types:

  • Use tissue microarrays containing multiple tissues to optimize simultaneously

  • Include known positive controls (specific for CK7) in each staining batch

  • Establish tissue-specific positivity thresholds (>10% for CK7 in CRC)

• Counterstaining considerations:

  • Adjust hematoxylin intensity to maintain visibility of membranous/cytoplasmic CK7 staining

  • Consider nuclear counterstains compatible with digital image analysis