MOK Antibody

What is MOK protein kinase and how is it characterized in antibody development?

MOK protein kinase (also known as RAGE-1, RAGE1, STK30, or renal cell carcinoma antigen) is a 419 amino acid residue protein (48 kDa) belonging to the CMGC Ser/Thr protein kinase family. It localizes in both the nucleus and cytoplasm, with up to six different isoforms reported . When developing or selecting antibodies against MOK:

• Target regions should consider the protein's domain structure

• Canonical epitopes are often located within amino acids 100-400

• Most commercial antibodies are raised against recombinant fragments or synthetic peptides

• Expression patterns (high in heart, brain, lung, kidney, and pancreas; low in placenta, liver, and skeletal muscle) should inform experimental controls

What applications are MOK antibodies most commonly used for?

MOK antibodies have demonstrated utility across multiple experimental applications, with varying success rates depending on epitope specificity and antibody format:

For optimal results, researchers should validate antibodies for their specific application, as performance varies significantly between applications even with the same antibody .

How should researchers optimize Western blot protocols for MOK detection?

For optimal Western blot detection of MOK protein:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors and phosphatase inhibitors if studying phosphorylated forms

  • Heat samples at 95°C for 5 minutes in reducing conditions

• Gel selection:

  • 10-12% SDS-PAGE gels are optimal for the 48 kDa MOK protein

  • Consider gradient gels (4-20%) when studying multiple isoforms

• Transfer conditions:

  • Semi-dry transfer: 15V for 30 minutes or wet transfer: 100V for 1 hour

  • PVDF membranes typically yield better results than nitrocellulose for MOK detection

• Blocking and antibody dilution:

  • 5% non-fat dry milk in TBST is generally effective

  • Most commercial MOK antibodies work optimally at 1:500-1:2000 dilution

  • Overnight incubation at 4°C often improves detection sensitivity

• Validation controls:

  • Include MOK-knockout or siRNA samples when possible

  • Positive controls should include tissues with known high expression (kidney, brain)

What are the recommended validation strategies for MOK antibodies?

Comprehensive validation of MOK antibodies should include:

• Western blot validation:

  • Verify the expected molecular weight (~48 kDa for canonical form)

  • Test specificity using genetic knockdown/knockout models

  • Verify absence of non-specific bands in tissues with low MOK expression

• Cross-reactivity testing:

  • Evaluate antibody performance across species of interest (human, mouse, rat, etc.)

  • BLAST analysis confirms high sequence identity among primates, rodents and some other vertebrates

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide to confirm specificity

  • Signal should be significantly reduced or eliminated

• Orthogonal validation:

  • Compare results from different antibodies targeting distinct MOK epitopes

  • Verify protein expression correlates with mRNA levels

• Application-specific validation:

  • For IHC: compare staining patterns with literature-reported distribution

  • For ChIP: verify enrichment at expected genomic loci

How can MOK antibodies be utilized in studying neuroinflammation mechanisms?

MOK plays a critical role in controlling inflammatory and type-I interferon responses in microglia. When designing experiments to study MOK in neuroinflammation:

• Experimental design considerations:

  • Use both in vitro microglial models (primary cultures or SIM-A9 cells) and in vivo CNS tissues

  • Include appropriate inflammatory stimuli (LPS, TDP-43 aggregates)

  • Compare MOK-knockout versus wild-type responses

• Key detection methodologies:

  • Immunofluorescence analysis of nuclear phospho-Brd4 (pBrd4) levels as a downstream marker of MOK activity

  • Western blot analysis of phosphorylated MOK (pThr159+pTyr161)

  • qRT-PCR assessment of IRF7 and other inflammatory genes regulated by MOK

• ChIP-qPCR approach:

  • Use MOK antibodies to assess binding to promoters of inflammatory genes

  • Focus on Il6, Ifnb1, and Tnfα gene promoters

  • Include pretreatment with MOK inhibitor (C13) as a control

  • Compare results between LPS-stimulated and unstimulated cells

• Analytical framework:

  • Correlate MOK activity with microglial morphology changes

  • Assess neuronal viability using conditioned media experiments

  • Monitor cytokine production as functional readouts

Research has shown that MOK levels are increased in ALS spinal cord samples, particularly in microglial cells, making it a valuable target for studying neuroinflammatory mechanisms in neurodegenerative diseases .

What methodological approaches are effective for studying MOK-Brd4 interactions?

The MOK-Brd4 interaction represents a key signaling axis in inflammatory responses. When investigating this pathway:

• Co-immunoprecipitation protocol:

  • Lyse cells in non-denaturing buffer with phosphatase inhibitors

  • Immunoprecipitate with anti-MOK antibody

  • Western blot for Brd4 and specifically pSer492-Brd4

  • Reverse IP with anti-Brd4 antibody can confirm interaction

• Phosphorylation analysis:

  • Use phospho-specific antibodies against Ser492-Brd4

  • Compare phosphorylation levels between wild-type and MOK-knockout cells

  • Include MOK inhibitor C13 as a control condition

• Chromatin occupancy assessment:

  • ChIP-qPCR using Brd4 antibodies on inflammatory gene promoters

  • Compare between MOK-knockout and wild-type cells

  • Use JQ1 (Brd4 inhibitor) as a control to confirm specificity

  • Focus on Il6, Ifnb1, and Tnfα promoter regions

• Confocal imaging approach:

  • Double immunofluorescence staining for MOK and Brd4

  • Quantify nuclear pBrd4 levels under different conditions

  • Track changes following LPS stimulation or TDP-43 aggregate exposure

The published data demonstrates that MOK regulates nuclear pBrd4 levels in microglia under inflammatory conditions, suggesting a direct regulatory relationship critical for inflammatory gene expression .

What are effective approaches for developing monoclonal antibodies against MOK protein?

Developing high-quality monoclonal antibodies against MOK requires strategic planning and rigorous characterization:

• Immunization strategies:

  • Consider both full recombinant proteins and synthetic peptides

  • Target conserved regions for broad species reactivity

  • For phospho-specific antibodies, use phosphopeptide immunogens

• Hybridoma generation and screening:

  • Encapsulate single cells into antibody capture hydrogels using droplet microfluidics for high-throughput screening

  • Use FACS to isolate antigen-specific antibody-secreting cells

  • Screen hybridoma supernatants initially by ELISA

• Binding characterization:

  • Assess binding affinity using surface plasmon resonance or ELISA

  • Determine epitope specificity through peptide mapping

  • Evaluate cross-reactivity with related kinases

• Functional validation:

  • Test antibody performance in multiple applications (WB, IHC, IF, IP)

  • Verify specificity using MOK-knockout or knockdown samples

  • Assess recognition of native versus denatured protein

• Sequencing and recombinant production:

  • Sequence immunoglobulin genes using Next Generation Sequencing

  • Express recombinant versions for consistent supply

  • This approach eliminates the need for long-term hybridoma maintenance

Modern antibody development techniques can facilitate rapid discovery, with reports of obtaining high-affinity monoclonal antibodies in as little as two weeks with high hit rates (>85% binding to target) .

How can MOK antibodies be utilized in multiplexed detection systems?

Multiplexed detection systems utilizing MOK antibodies can enhance experimental throughput and data acquisition:

• Multicolored nanoparticle approach:

  • Conjugate MOK antibodies to differently colored gold nanoparticles (red nanospheres or blue nanostars)

  • Use in immunochromatography formats for multiplexed detection

  • Analyze signal patterns using RGB color component analysis and principal component analysis

• Multiplex immunoassay design:

  • Combine MOK antibodies with antibodies against related signaling molecules

  • Use antibody pairs recognizing different epitopes to enhance specificity

  • Implement machine learning for deconvolution of complex signals

• Simoa Planar Array optimization:

  • Optimize capture and detection antibody concentrations

  • Design full factorial experimental design to reduce experimental effort

  • This approach has achieved femtomolar detection limits for immunoglobulins

• Validation considerations:

  • Include appropriate positive and negative controls

  • Assess cross-reactivity between detection antibodies

  • Evaluate signal interference in complex biological samples

These multiplexed approaches can be particularly valuable when studying MOK in relation to inflammatory signaling networks, allowing simultaneous detection of multiple components of the pathway .

How can researchers effectively use phospho-specific MOK antibodies?

Phospho-specific MOK antibodies are crucial for studying its activation state and signaling functions:

• Key phosphorylation sites:

  • pThr159+pTyr161 are critical for MOK activation

  • Phospho-specific antibodies against these sites are commercially available

• Sample preparation for phospho-detection:

  • Immediate sample processing is crucial to preserve phosphorylation status

  • Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • Maintain samples at 4°C throughout processing

• Assay optimization for phospho-MOK detection:

  • For Western blots: use 5% BSA instead of milk for blocking

  • For immunoprecipitation: adjust lysis buffers to preserve phosphorylation

  • For IHC: consider antigen retrieval methods that preserve phospho-epitopes

• Experimental controls:

  • Include phosphatase-treated samples as negative controls

  • Use MOK inhibitor (C13) treatment as an additional control

  • Compare stimulated vs. unstimulated samples to verify dynamic changes

• Signaling pathway analysis:

  • Monitor downstream effectors like pBrd4 to confirm functional significance

  • Assess correlation between MOK phosphorylation and microglial activation

  • Compare phosphorylation patterns in disease models versus controls

Studies have shown increased phospho-MOK levels in activated microglia in ALS models, suggesting its potential as a biomarker for neuroinflammation .

What are the methodological considerations for using MOK antibodies in chromatin immunoprecipitation (ChIP) studies?

ChIP experiments with MOK antibodies require careful optimization:

• ChIP protocol optimization:

  • Crosslinking: Use 1% formaldehyde for 10 minutes at room temperature

  • Sonication: Optimize conditions to generate 200-500bp DNA fragments

  • Antibody selection: Choose antibodies validated for ChIP applications

• Control strategies:

  • Include IgG negative controls

  • Use MOK-knockout cells as additional negative controls

  • Perform MOK inhibitor (C13) treatment as a functional control

• MOK-Brd4 co-localization analysis:

  • Perform sequential ChIP (Re-ChIP) to detect co-occupancy

  • Compare binding patterns at inflammatory gene promoters

  • Focus on Il6, Ifnb1, and Tnfα promoter regions

• Data analysis approaches:

  • Normalize enrichment to input samples

  • Compare binding between stimulated and unstimulated conditions

  • Correlate MOK binding with transcriptional activity

• Integration with other approaches:

  • Combine with RNA-Seq data to correlate binding with expression

  • Use ATAC-Seq to assess chromatin accessibility at binding sites

  • Perform phospho-MOK ChIP to determine if phosphorylation affects binding

Research has demonstrated that MOK regulates Brd4 binding to inflammatory gene promoters, highlighting the importance of this approach for understanding MOK's role in transcriptional regulation .