CK10 is a type II cytokeratin expressed in suprabasal keratinocytes of stratified squamous epithelia, including skin and mucosal tissues . It forms heterodimers with cytokeratin 9 (CK9) to stabilize epithelial cell layers and maintain barrier function .
Below are widely used CK10 antibodies, validated across species and applications:
CK10 antibodies are critical for studying skin barrier integrity:
Plantar Skin: CK10 is essential for establishing the epidermal barrier in plantar skin .
Microbial Adhesion: CK10 mediates bacterial adherence via interactions with Staphylococcus aureus (clfB) and Streptococcus pneumoniae (PsrP) .
Carcinoma Detection: CK10 antibodies distinguish squamous cell carcinomas from adenocarcinomas in IHC .
Viral Infection Models: CK10 expression patterns inform studies on epithelial responses to pathogens .
Western Blot: EP1607IHCY detects a 60 kDa band in human A431 and HaCaT cell lysates .
IHC Optimization: Heat-mediated antigen retrieval (Tris/EDTA, pH 9) enhances CK10 staining in paraffin sections .
CRK10 Kinase Antibodies: No CRK10-specific antibodies are documented in the provided sources; studies on Arabidopsis CRK10 focus on genetic mutants (e.g., crk10-A397T), not immunodetection .
Species Specificity: CK10 antibodies vary in cross-reactivity; RKSE60 shows broader species compatibility (zebrafish, pig) .
CRK10 (Cysteine-rich receptor-like kinase 10) is a receptor-like kinase in Arabidopsis that plays a significant role in regulating xylem vessel development. The protein contains a kinase domain with critical regulatory regions, including the αC helix where mutations can significantly alter its function. Research indicates that CRK10 is involved in plant development pathways, particularly in vascular tissue formation, as evidenced by abnormal xylem vessel phenotypes observed in mutants like crk10-A397T .
When investigating CRK10 with antibodies, researchers should consider its expression patterns in different tissues and developmental stages. Most effective antibodies for CRK10 detection would recognize conserved epitopes in the kinase domain or specific regions that distinguish it from other CRK family members.
For optimal western blot detection of CRK10:
Sample preparation: Extract proteins using TRI Reagent or similar buffer systems that effectively solubilize membrane-associated proteins.
Protein loading: Load 20-50 μg of total protein per lane.
Separation: Use 10-12% SDS-PAGE gels for effective separation.
Transfer: Employ PVDF membranes (0.45 μm pore size) with semi-dry transfer at 25V for 30 minutes.
Blocking: Block with 5% non-fat dry milk in TBST for 1 hour at room temperature.
Primary antibody: Dilute CRK10 antibody at 1:1000 to 1:2000 in blocking buffer and incubate overnight at 4°C.
Detection: Use HRP-conjugated secondary antibodies with appropriate chemiluminescent detection systems.
For validation, consider using recombinant CRK10 protein domains as positive controls, similar to the approach used for expression and purification of CRK10 kinase domain described in the literature .
Based on established protocols for CRK10 research, the most reliable method for quantifying CRK10 expression is quantitative PCR (qPCR):
RNA isolation: Extract total RNA using TRI Reagent from target tissues.
DNase treatment: Treat RNA with DNase I (Amplification Grade) to remove genomic DNA contamination.
cDNA synthesis: Use SuperScript III Reverse Transcriptase or equivalent enzymes.
qPCR setup: Perform reactions using FastStart Essential DNA Green Master or similar SYBR Green-based systems.
Reference genes: Normalize expression using stable reference genes such as ACTIN2 (ACT2) and UBC21.
Data analysis: Calculate relative expression using the 2^(-ΔΔCt) method .
For protein-level quantification using CRK10 antibodies, standard curves generated with recombinant CRK10 protein can provide absolute quantification when performing immunoblotting or ELISA assays.
To validate CRK10 antibody specificity:
Genetic controls:
Compare antibody reactivity between wild-type and crk10 knockout/knockdown lines
Use overexpression lines (35S:CRK10) as positive controls
Biochemical validation:
Pre-adsorption tests using recombinant CRK10 protein
Competition assays with increasing concentrations of purified CRK10
Western blot analysis comparing predicted versus observed molecular weight
Cross-reactivity assessment:
Test reactivity against related CRK family members
Examine potential cross-reactivity with the kinase domains of similar receptor-like kinases
Recombinant protein controls:
When conducting immunolocalization studies with CRK10 antibodies:
Genetic controls:
Technical controls:
Pre-immune serum application (background control)
Primary antibody omission
Secondary antibody only controls
Peptide competition assays
Validation approaches:
Tissue-specific considerations:
Based on available data, focus on vascular tissues where CRK10 plays critical roles in xylem vessel development
For investigating CRK10 protein interactions:
Co-immunoprecipitation (Co-IP):
Use purified CRK10 antibodies conjugated to agarose/magnetic beads
Extract proteins under native conditions to preserve interactions
Validate interactions through reciprocal Co-IPs with antibodies against suspected interacting partners
Consider mild detergents (0.5-1% NP-40 or Digitonin) to maintain membrane protein interactions
Proximity ligation assay (PLA):
Apply CRK10 antibodies in combination with antibodies against potential interacting proteins
Optimize fixation methods to preserve protein complexes while maintaining epitope accessibility
Use appropriate positive controls (known interacting proteins) and negative controls
Pull-down validation:
Post-translational modifications (PTMs) can significantly impact antibody recognition of CRK10:
Characterization strategies:
Compare antibody reactivity under different physiological conditions that may alter PTMs
Use phosphatase treatment to remove phosphorylation prior to western blotting
Apply glycosidase treatments to remove N-linked or O-linked glycans
Epitope-specific considerations:
Generate phospho-specific antibodies targeting known phosphorylation sites
Consider antibodies recognizing different domains (extracellular, kinase, C-terminal)
Mutation analysis:
MS validation approach:
Use mass spectrometry to identify PTMs on immunoprecipitated CRK10
Compare PTM patterns in different experimental conditions
To effectively analyze CRK10 kinase activity:
Experimental design for phospho-specific detection:
In vitro kinase assays:
Express and purify recombinant CRK10 kinase domain
Perform assays with γ-32P-ATP or ATP and phospho-specific antibodies
Compare activities between wild-type and mutant variants
Inhibitor studies:
Use specific kinase inhibitors to validate phosphorylation specificity
Include appropriate controls with inhibitors of related kinases
Signaling pathway analysis:
Monitor downstream phosphorylation events using phospho-specific antibodies
Compare signaling outputs between wild-type plants and crk10 mutants
Inconsistent results with CRK10 antibodies across tissues may result from:
Expression level variations:
Extraction method limitations:
Membrane-associated proteins like CRK10 require specific extraction protocols
Different tissues may require adjusted extraction buffers
Solution: Optimize extraction methods for each tissue type (consider detergent types/concentrations)
Post-translational modifications:
Tissue-specific PTMs may alter epitope accessibility
Solution: Use multiple antibodies targeting different regions of CRK10
Cross-reactivity issues:
Related CRK family members may be expressed at different levels across tissues
Solution: Validate specificity in each tissue context using genetic controls
For detecting low-abundance CRK10:
Sample enrichment techniques:
Immunoprecipitation prior to western blotting
Subcellular fractionation to concentrate membrane fractions
Use of tissue-specific promoters to isolate relevant cell types
Signal amplification methods:
TSA (Tyramide Signal Amplification) for immunohistochemistry
Enhanced chemiluminescence substrates for western blot
Consider more sensitive detection systems like Wes™ or Jess™ automated western blotting
Genetic approaches:
Optimization table for low-abundance detection:
Method | Sample Amount | Antibody Dilution | Incubation | Detection System |
---|---|---|---|---|
Standard WB | 50-100 μg | 1:1000 | Overnight, 4°C | Standard ECL |
IP-WB | 500-1000 μg | 1:2000 | 2 hr, RT | Enhanced ECL |
IHC | Sections | 1:100 | Overnight, 4°C | TSA amplification |
Automated WB | 10-20 μg | 1:50 | System default | Chemiluminescence |
To distinguish specific from non-specific binding:
Critical validation controls:
Genetic knockout/knockdown lines (crk10 mutants)
Pre-absorption of antibody with recombinant CRK10 protein
Comparison with CRK10-tagged protein expression patterns
Technical optimization:
Titrate antibody concentrations to determine optimal signal-to-noise ratio
Optimize blocking conditions (test BSA vs. milk vs. commercial blockers)
Adjust washing stringency and duration
Cross-reactivity analysis:
Test antibody against recombinant proteins of related CRK family members
Use peptide competition with both specific and non-specific peptides
Analysis methods:
Compare banding patterns with predicted molecular weights
Assess pattern changes under different experimental conditions
For high-throughput applications with CRK10 antibodies:
Assay adaptation strategies:
Develop ELISA-based methods for CRK10 quantification
Create multiplex assays combining CRK10 detection with other signaling components
Adapt to automated western blot systems for consistent results
Screening optimization:
Miniaturize immunoassays for microplate formats
Develop homogeneous assay formats (no-wash steps)
Create reporter cell lines expressing CRK10 with activity-dependent readouts
Validation considerations:
Establish Z-factor and signal window for assay quality control
Include appropriate controls on each plate
Validate hits with orthogonal methods
Automated imaging applications:
Adapt immunofluorescence protocols for high-content imaging
Develop automated image analysis algorithms for quantification
Advanced methodological approaches for specific CRK10 detection:
Parallel reaction monitoring (PRM):
Combine immunoprecipitation with targeted mass spectrometry
Develop specific peptide transitions for CRK10 detection
Enables absolute quantification with isotope-labeled standards
Proximity-based detection:
Develop split reporter systems (split GFP, split luciferase) with CRK10
Apply proximity extension assays for sensitive detection
Use proximity ligation for in situ detection of protein complexes
CRISPR-based tagging:
Endogenously tag CRK10 with epitope tags or fluorescent proteins
Preserves native expression levels and regulation
Enables direct visualization and quantification
Computational approaches:
Develop machine learning algorithms for improved image analysis
Create prediction tools for antibody-epitope interactions
Model CRK10 structure to identify optimal epitopes for antibody generation
When investigating CRK10 mutant variants (such as the A397T mutation described in the search results):
Antibody selection considerations:
Determine if mutations affect epitope recognition
Use multiple antibodies targeting different regions
Consider generating mutation-specific antibodies
Control design:
Functional assessment approaches:
Compare phosphorylation states between wild-type and mutant variants
Assess protein-protein interactions and how mutations affect them
Quantify downstream signaling outputs
Data integration strategy: