CDKL2 Antibody

What is CDKL2 and what are its primary functions in cellular biology?

CDKL2 (Cyclin-dependent kinase-like 2) is a serine/threonine protein kinase belonging to the CDKL family. It's also known as p56 KKIAMRE protein kinase. CDKL2 functions as a regulator of epithelial-mesenchymal transition (EMT) in cancer cells, particularly in breast cancer. Research has demonstrated its involvement in cellular signaling pathways related to cell proliferation and potentially cancer progression. Studies indicate it may have distinct roles in different cancer types, including gastric cancer where it appears to interact with HER2 signaling pathways .

What types of CDKL2 antibodies are currently available for research applications?

There are several types of validated CDKL2 antibodies available for research:

Most antibodies are unconjugated but can be paired with appropriate secondary antibodies depending on experimental design requirements .

What are the most common applications for CDKL2 antibodies in cancer research?

CDKL2 antibodies are primarily used in cancer research for:

• Western Blot (WB): Detecting CDKL2 protein expression levels in cell or tissue lysates, typically visualized at approximately 56 kDa molecular weight

• Immunohistochemistry (IHC): Evaluating CDKL2 expression patterns in tissue sections, particularly useful in cancer tissue microarrays

• Immunofluorescence (IF): Determining subcellular localization of CDKL2 in intact cells

• ELISA: Quantitative measurement of CDKL2 protein levels in biological samples

How can I properly validate a CDKL2 antibody for my specific research application?

Comprehensive CDKL2 antibody validation should include:

• Positive and negative controls: Use cell lines with known CDKL2 expression levels. HEK293T cells transfected with CDKL2 expression vectors can serve as positive controls .

• Cross-reactivity testing: If working with non-human samples, verify species cross-reactivity with appropriate controls.

• Knockdown/knockout validation: Use siRNA or CRISPR to reduce/eliminate CDKL2 expression, then confirm antibody signal reduction.

• Application-specific validation:

  • For WB: Confirm single band at expected molecular weight (~56 kDa)

  • For IHC: Compare staining patterns with mRNA expression data

  • For IF: Verify subcellular localization consistent with known biology

• Epitope specificity: When using antibodies recognizing specific domains (like N-terminal antibodies), confirm specificity with competing peptides .

• Multiple antibody approach: Use two different antibodies targeting different epitopes to confirm findings.

For Mouse-derived antibodies used on mouse tissues, consider using Mouse-On-Mouse blocking reagents to reduce background signal .

What are the key considerations when designing CDKL2 protein detection experiments in relation to HER2 status?

When investigating CDKL2 in relation to HER2 status:

• Sample selection: Include tissues/cells with known HER2 status (positive and negative) to establish baseline correlations.

• Combined methodological approach: Follow established protocols like those used in recent GC studies :

  • Detect CDKL2 protein expression by IHC (calculating H-scores considering both staining extent and intensity)

  • Determine CDKL2 gene copy number by FISH

  • Correlate both with HER2 status determined by standardized criteria

• Statistical analysis: Employ multivariate Cox proportional hazard regression analysis to evaluate whether CDKL2 is an independent marker or correlates with HER2 status.

• Control for heterogeneity: When calculating H-scores, weight different staining intensity scores by their corresponding extent scores to account for heterogeneous staining patterns .

• Combined protein and genetic analysis: Analyze both CDKL2 protein expression and gene copy number for comprehensive assessment, as research indicates they are significantly correlated .

How should I interpret contradictory findings regarding CDKL2 expression patterns across different cancer types?

When facing contradictory findings about CDKL2's role:

What are the optimal protocols for CDKL2 immunohistochemistry on paraffin-embedded tissue sections?

For optimal CDKL2 IHC on FFPE tissues:

What are the critical steps for successful Western blot detection of CDKL2?

For reliable Western blot detection of CDKL2:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for cell/tissue lysis

  • Include phosphatase inhibitors if phosphorylation status is relevant

  • Ensure equal protein loading (20-50μg total protein per lane)

• Gel selection and transfer:

  • Use 10% SDS-PAGE gels for optimal CDKL2 separation (MW ~56 kDa)

  • Transfer to PVDF membranes (preferred over nitrocellulose for this protein)

• Blocking and antibody dilutions:

  • Block with 5% non-fat dry milk or BSA in TBST

  • Primary antibody dilutions:

    • Mouse monoclonal: 1:500-1:2000

    • Rabbit polyclonal: 1:500-1:1000

  • Incubate overnight at 4°C for optimal results

• Visualization strategy:

  • Use HRP-conjugated secondary antibodies with enhanced chemiluminescence

  • Expected band size: 56 kDa (theoretical MW 55.8 kDa)

• Controls and validation:

  • Positive control: HEK293T cells transfected with CDKL2 expression vector

  • Negative control: Non-transfected cells or cells with CDKL2 knockdown

  • Consider running Jurkat cell lysates as they show detectable endogenous CDKL2 expression

How can I optimize immunofluorescence protocols for CDKL2 subcellular localization studies?

For optimal CDKL2 immunofluorescence:

• Cell preparation:

  • Culture cells on glass coverslips or chamber slides

  • Fix with 4% paraformaldehyde (10 minutes, room temperature)

  • Permeabilize with 0.2% Triton X-100 (5 minutes)

• Blocking and antibody conditions:

  • Block with 3-5% BSA or normal serum from secondary antibody host species

  • Primary antibody dilutions:

    • Mouse monoclonal antibodies: 1:100-1:200

    • Rabbit polyclonal antibodies: 1:100-1:500

  • Incubate overnight at 4°C in humidified chamber

• Multiple labeling strategy:

  • Combine CDKL2 staining with markers for subcellular compartments:

    • Nuclear markers (DAPI)

    • Cytoskeletal markers (α-tubulin)

    • Membrane markers as appropriate

• Image acquisition and analysis:

  • Use confocal microscopy for precise subcellular localization

  • Analyze colocalization with appropriate software (ImageJ with colocalization plugins)

  • Quantify nuclear/cytoplasmic distribution ratios

• Controls:

  • Include cells with CDKL2 overexpression or knockdown

  • Perform secondary-only controls to assess background

  • For mouse antibodies on mouse cells, use Mouse-On-Mouse blocking reagents

How should I interpret correlations between CDKL2 expression, gene copy number, and clinical outcomes?

When analyzing CDKL2 expression, gene copy number, and clinical data:

How can I reconcile contradictory findings between CDKL2 mRNA, protein expression, and gene copy number data?

To resolve apparent contradictions between different molecular data types:

• Multi-layer data integration strategies:

  • Compare protein levels (by IHC/WB) with mRNA expression (by qRT-PCR/RNA-seq)

  • Correlate both with gene copy number alterations (FISH/CGH)

  • Consider post-transcriptional and post-translational regulation

• Methodological considerations:

  • Antibody specificity: Ensure antibodies detect the appropriate isoforms

  • Sample matching: Use matched samples for different analyses when possible

  • Technical validation: Use multiple techniques to confirm findings

• Biological interpretation frameworks:

  • Protein levels often correlate poorly with mRNA due to post-transcriptional regulation

  • Gene amplification doesn't always result in proportional protein overexpression

  • Consider tumor heterogeneity within samples

• Context-specific analysis:

  • Different tissues/cancer types may show different CDKL2 regulation patterns

  • Examine differential expression in subgroups based on clinical parameters

• Functional validation approach:

  • Perform in vitro studies to validate the functional consequences of altered CDKL2 expression

  • Use cell models with CDKL2 overexpression or knockdown to validate observations

What are the key considerations when designing experiments to explore CDKL2's role in epithelial-mesenchymal transition?

For investigating CDKL2's role in EMT:

• Cell model selection:

  • Use epithelial cancer cell lines known to undergo EMT

  • Include both CDKL2-high and CDKL2-low cell lines

  • Consider paired primary/metastatic cell lines

• Experimental manipulation approaches:

  • Overexpression: Transfect cells with CDKL2 expression vectors

  • Knockdown: Use siRNA or shRNA targeting CDKL2

  • Knockout: CRISPR/Cas9 system for complete gene deletion

  • Pharmacological: If available, use specific CDKL2 kinase inhibitors

• EMT marker assessment:

  • Epithelial markers: E-cadherin, ZO-1, claudins

  • Mesenchymal markers: N-cadherin, vimentin, fibronectin

  • EMT transcription factors: Snail, Slug, ZEB1/2, Twist

  • Monitor changes by WB, qRT-PCR, and immunofluorescence

• Functional assays:

  • Migration: Wound healing/scratch assays

  • Invasion: Transwell invasion assays

  • Morphological changes: Phase contrast microscopy

  • Cell-cell adhesion: Aggregation assays

• Signaling pathway analysis:

  • Investigate interaction with HER2 signaling based on recent findings

  • Examine MAPK, PI3K/AKT, and Wnt pathways commonly involved in EMT

  • Use phospho-specific antibodies to assess activation status

• In vivo validation:

  • Xenograft models with CDKL2-modified cells

  • Analyze metastatic potential and correlation with EMT markers

  • Consider patient-derived xenografts for clinical relevance

What are the emerging applications of CDKL2 antibodies in cancer biomarker development?

Emerging applications for CDKL2 antibodies in biomarker research include:

• Combined diagnostic approaches:

  • Development of multi-marker panels combining CDKL2 with HER2 and other markers

  • Integration into prognostic algorithms for gastric and other cancers

  • Application in multiplex IHC/IF platforms for simultaneous detection with other markers

• Liquid biopsy development:

  • Detection of circulating CDKL2 protein in patient serum

  • Correlation with tissue expression and disease progression

  • Development of highly sensitive ELISA or other immunoassays for CDKL2

• Therapy response prediction:

  • Monitoring CDKL2 expression before and during treatment

  • Correlation with response to HER2-targeted therapies

  • Potential application in guiding treatment decisions

• Advanced imaging applications:

  • Development of CDKL2-targeting antibodies for in vivo imaging

  • Fluorescent or radiolabeled derivatives for cancer detection

  • Intraoperative imaging to guide surgical resection

• Therapeutic target potential:

  • Investigation of CDKL2 as a druggable target in cancer

  • Development of function-blocking antibodies

  • Antibody-drug conjugates targeting CDKL2-expressing cells

Current evidence suggests CDKL2 detection could have particular value in predicting HER2 status and prognosis in gastric cancer patients, potentially improving treatment stratification .

How can single-cell analysis techniques be combined with CDKL2 antibodies to advance cancer research?

Integration of CDKL2 antibodies with single-cell technologies:

• Single-cell proteomics approaches:

  • Mass cytometry (CyTOF) incorporating CDKL2 antibodies into panels

  • Single-cell Western blotting for CDKL2 detection

  • Microfluidic antibody capture for quantitative single-cell protein analysis

• Spatial transcriptomics integration:

  • Combining CDKL2 IHC with spatial transcriptomics

  • Correlating protein localization with mRNA expression patterns

  • Mapping CDKL2 expression in tumor microenvironment context

• Single-cell multi-omics strategies:

  • CITE-seq approaches incorporating CDKL2 antibodies

  • Simultaneous detection of CDKL2 protein and mRNA in single cells

  • Correlation with genomic features at single-cell resolution

• Tumor heterogeneity assessment:

  • Quantifying cell-to-cell variation in CDKL2 expression

  • Identifying rare CDKL2-high subpopulations with distinct properties

  • Correlating with other cancer stem cell or EMT markers

• Technological considerations:

  • Antibody conjugation to appropriate tags for single-cell methods

  • Validation of specificity in highly sensitive single-cell applications

  • Development of computational pipelines for integrated analysis

These approaches could help resolve contradictory findings by revealing differential CDKL2 expression patterns in specific cell populations within heterogeneous tumors.

What experimental approaches should be considered to explore the potential of CDKL2 as a therapeutic target?

Evaluating CDKL2 as a therapeutic target requires:

• Target validation strategies:

  • Comprehensive expression profiling across normal and cancerous tissues

  • Genetic dependency screening (CRISPR, shRNA) to identify CDKL2-dependent cancers

  • In vivo validation using conditional knockout models

• Structural and functional characterization:

  • Crystal structure determination of CDKL2 kinase domain

  • Identification of key substrate proteins and phosphorylation sites

  • Mapping of interaction partners in relevant cancer types

• Drug development approaches:

  • Small molecule inhibitor development targeting CDKL2 kinase activity

  • Structure-based drug design using computational modeling

  • High-throughput screening of compound libraries

• Antibody-based therapeutic strategies:

  • Development of function-blocking antibodies

  • Antibody-drug conjugates targeting CDKL2-expressing cells

  • Bispecific antibodies targeting CDKL2 and other cancer markers

• Combination therapy exploration:

  • Testing with HER2-targeted therapies based on correlation findings

  • Synergy studies with conventional chemotherapeutics

  • Combination with immune checkpoint inhibitors

• Biomarker development for patient selection:

  • Standardized IHC protocols for patient stratification

  • Combined gene copy number and protein expression assessment

  • Development of companion diagnostics for CDKL2-targeted therapies

The tight correlation between CDKL2 and HER2 status suggests potential value in combination approaches targeting both pathways simultaneously .