CRK10 Antibody

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Description

Cytokeratin 10 (CK10) Antibodies: Overview

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 .

Key Applications of CK10 Antibodies:

ApplicationMethodKey Use Cases
Immunohistochemistry (IHC)Paraffin-embedded tissue stainingDiagnosing squamous differentiation in carcinomas
Western Blot (WB)Protein expression profilingAnalyzing CK10 levels in skin lysates
Immunocytochemistry (ICC)/IFCellular localization studiesVisualizing CK10 in keratinocyte cultures
Flow CytometryEpithelial cell identificationCharacterizing CK10+ cell populations

Prominent CK10 Antibody Clones and Products

Below are widely used CK10 antibodies, validated across species and applications:

Epithelial Barrier Function

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) .

Diagnostic and Prognostic Utility

  • 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 .

Technical Validation

  • 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 .

Table 2: Performance Across Applications

Antibody CloneWB SensitivityIHC SpecificityCross-Reactivity
EP1607IHCY1:10,000–1:50,000 High (multi-TMA validated) Human, Mouse, Rat
DE-K10UndisclosedModerateHuman, Canine, Rat
RKSE60UndisclosedHigh (native CK10 immunogen) Human, Zebrafish, Pig

Limitations and Considerations

  • 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) .

Product Specs

Buffer
Preservative: 0.03% Proclin 300; Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Form
Liquid
Lead Time
14-16 week lead time (made-to-order)
Synonyms
CRK10 antibody; RLK4 antibody; At4g23180 antibody; F21P8.70Cysteine-rich receptor-like protein kinase 10 antibody; Cysteine-rich RLK10 antibody; EC 2.7.11.- antibody; Receptor-like protein kinase 4 antibody
Target Names
CRK10
Uniprot No.

Target Background

Database Links

KEGG: ath:AT4G23180

STRING: 3702.AT4G23180.1

UniGene: At.19078

Protein Families
Protein kinase superfamily, Ser/Thr protein kinase family, CRK subfamily
Subcellular Location
Membrane; Single-pass membrane protein.

Q&A

What is CRK10 and what cellular functions does it regulate?

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.

How should I optimize western blot protocols for CRK10 antibody detection?

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 .

What are the best methods for quantifying CRK10 expression in plant tissues?

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.

How can I design experiments to validate CRK10 antibody specificity?

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:

    • Generate His-tagged CRK10 kinase domain constructs similar to those described in the literature

    • Include both wild-type and mutant versions (e.g., A397T and D473N variants) as controls

What are the appropriate experimental controls for CRK10 immunolocalization studies?

When conducting immunolocalization studies with CRK10 antibodies:

  • Genetic controls:

    • CRK10 knockout/knockdown lines (negative control)

    • CRK10-mCherry translational fusion lines (positive control reference)

    • CRK10 overexpression lines (35S:CRK10)

  • Technical controls:

    • Pre-immune serum application (background control)

    • Primary antibody omission

    • Secondary antibody only controls

    • Peptide competition assays

  • Validation approaches:

    • Compare immunolocalization with GFP/mCherry fusion protein localization

    • Correlate with CRK10 promoter-driven reporter expression patterns (CRK10 Pro:GUS)

    • Verify cellular compartment with co-localization using organelle markers

  • Tissue-specific considerations:

    • Based on available data, focus on vascular tissues where CRK10 plays critical roles in xylem vessel development

How can CRK10 antibodies be used to investigate protein-protein interactions?

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:

    • Express recombinant CRK10 kinase domain constructs (His-tagged KD) similar to those described in the literature

    • Use for affinity purification of interacting partners

    • Compare wild-type and mutant versions (A397T and D473N) to understand how mutations affect interactions

What approaches help determine if post-translational modifications affect CRK10 antibody recognition?

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:

    • Compare antibody reactivity between wild-type CRK10 and mutant variants (e.g., A397T)

    • Assess how mutations may change conformation and expose/hide epitopes

  • MS validation approach:

    • Use mass spectrometry to identify PTMs on immunoprecipitated CRK10

    • Compare PTM patterns in different experimental conditions

How should I design experiments to analyze CRK10 activity using phosphorylation-specific antibodies?

To effectively analyze CRK10 kinase activity:

  • Experimental design for phospho-specific detection:

    • Generate or obtain phospho-specific antibodies targeting known autophosphorylation sites

    • Compare phosphorylation levels between wild-type and kinase-dead (D473N) variants

    • Assess how gain-of-function mutations (A397T) affect phosphorylation status

  • 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

Why might I observe inconsistent results with CRK10 antibodies in different tissue types?

Inconsistent results with CRK10 antibodies across tissues may result from:

  • Expression level variations:

    • CRK10 expression can vary significantly between tissues and developmental stages

    • qPCR analysis shows tissue-specific expression patterns that should correlate with protein detection

    • Solution: Adjust antibody concentrations based on expected expression levels

  • 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

What strategies can overcome challenges in detecting low-abundance CRK10 protein?

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:

    • Generate transgenic lines with epitope-tagged CRK10 under native promoter

    • Use CRK10-fluorescent protein fusions for direct visualization

  • Optimization table for low-abundance detection:

MethodSample AmountAntibody DilutionIncubationDetection System
Standard WB50-100 μg1:1000Overnight, 4°CStandard ECL
IP-WB500-1000 μg1:20002 hr, RTEnhanced ECL
IHCSections1:100Overnight, 4°CTSA amplification
Automated WB10-20 μg1:50System defaultChemiluminescence

How can I distinguish between specific and non-specific binding of CRK10 antibodies?

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

How can CRK10 antibodies be adapted for high-throughput screening applications?

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

What methodological advances improve CRK10 detection specificity in complex protein mixtures?

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

How should experimental design be modified when investigating CRK10 mutant variants?

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:

    • Include both wild-type and kinase-dead (D473N) variants

    • Generate recombinant proteins of each variant for validation

    • Create transgenic lines expressing different variants at comparable levels

  • 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:

    • Correlate phenotypic outcomes with molecular changes

    • Connect protein-level analyses with observed physiological effects

    • Examine how mutations in CRK10 (like A397T) affect xylem vessel development

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