CSNK1G3 Antibody, Biotin conjugated

What is CSNK1G3 and what cellular functions does it regulate?

CSNK1G3 (Casein kinase I isoform gamma-3) is a serine/threonine-protein kinase that preferentially phosphorylates acidic proteins such as caseins. This enzyme participates in several critical cellular processes:

• Wnt signaling pathway regulation: CSNK1G3 plays a unique role in activating β-catenin-dependent WNT signaling compared to other family members

• Oxidative stress response: Recent research identified CSNK1G3 as a regulator of oxidative stress response and reactive oxygen species (ROS) levels through interactions with the NADPH dual oxidase complex

• Synaptic transmission: CSNK1G3 regulates fast synaptic transmission mediated by glutamate

• Protein phosphorylation: It can phosphorylate numerous proteins in various cellular contexts

The protein contains a highly conserved kinase domain and a unique C-terminal regulatory domain with a 33 amino acid insertion not found in other family members .

How does biotin conjugation affect CSNK1G3 antibody applications?

Biotin conjugation provides several methodological advantages when working with CSNK1G3 antibodies:

• Enhanced detection sensitivity: The biotin-streptavidin system provides signal amplification in detection methods due to the high affinity interaction (Kd ≈ 10^-15 M)

• Versatile detection options: Allows flexible detection using streptavidin conjugated to various reporter molecules (HRP, fluorophores, gold particles)

• Application compatibility: Biotin-conjugated antibodies are particularly valuable for ELISA, immunohistochemistry, and detection systems requiring signal amplification

• Multi-layered detection systems: Useful in protocols requiring sequential antibody applications or when trying to minimize background signal

When selecting between conjugated and unconjugated CSNK1G3 antibodies, consider your specific detection requirements and whether direct detection or a multi-step protocol best suits your experimental design.

What experimental applications are suitable for CSNK1G3 Antibody, Biotin conjugated?

The CSNK1G3 Antibody, Biotin conjugated has been validated for several research applications:

The antibody has been affinity-purified using recombinant Human Casein kinase I isoform gamma-3 protein (amino acids 1-204) as the immunogen, making it highly specific for CSNK1G3 .

How should I design experiments to study CSNK1G3's unique role in WNT signaling?

When investigating CSNK1G3's function in WNT signaling, consider these methodological approaches:

• Comparative analysis with other family members: Research has shown that only CSNK1G3, not CSNK1G1 or CSNK1G2, activates β-catenin-dependent WNT signaling when overexpressed . Design experiments that compare all three family members.

• LRP6 phosphorylation analysis: CSNK1G3 uniquely induces LRP6 phosphorylation at both T1479 and S1490 sites, while CSNK1G1 shows no effect and CSNK1G2 only affects S1490 phosphorylation in response to WNT3A . Include phospho-specific antibodies in your analysis.

• Interdependency studies: Experimental data indicates that:

  • WNT ligand secretion (test using PORCN inhibitor C59)

  • LRP5/6 expression (verified in LRP5/6 knockout cells)

  • DVL proteins (partial requirement)

  are all involved in CSNK1G3-driven WNT activation .

• Dose-response overexpression: In contrast to other family members, CSNK1G3 dose-dependently activates WNT reporter (BAR) systems .

What controls should I include when using CSNK1G3 Antibody, Biotin conjugated for immunodetection?

To ensure reliable data when using CSNK1G3 Antibody, Biotin conjugated, include these essential controls:

• Positive control samples:

  • HeLa cell lysates (verified to express endogenous CSNK1G3)

  • Recombinant CSNK1G3 protein (particularly the 1-204AA region used as immunogen)

• Negative controls:

  • CSNK1G3 knockout cell lysates (can be generated using protocols in source )

  • Sp2/0 cell lysate (shown as negative control in validation data)

  • Secondary detection reagent only (no primary antibody)

• Specificity controls:

  • Pre-incubation with blocking peptide derived from CSNK1G3 N-terminal region

  • Comparison with other CSNK1G3 antibodies recognizing different epitopes

  • Analysis of cross-reactivity with CSNK1G1 and CSNK1G2 when relevant

• Endogenous biotin blocking:

  • When working with tissues known to contain endogenous biotin (liver, kidney, brain), use avidin/biotin blocking steps prior to antibody application

How can I validate CSNK1G3 knockout or gene editing in my cellular models?

To confirm successful CSNK1G3 knockout or gene editing, employ multiple validation methods:

• Genomic validation:

  • PCR amplification and sequencing of the targeted region

  • For C-terminal modifications, focus on exon 12 which encodes the C-terminal region

• Protein level validation:

  • Western blot analysis using antibodies against different epitopes of CSNK1G3

  • Immunocytochemistry to confirm absence or modification of protein expression

• Functional validation:

  • Lysenin resistance assay: C-terminally truncated CSNK1G3 mutants exhibit lysenin resistance, while wild-type and complete knockout cells remain sensitive

  • MTT assay protocol for lysenin resistance:

    • Seed cells at 2.5 × 10^4 cells/well in a 12-well plate

    • Culture 18-24h at 37°C

    • Wash with serum-free medium

    • Add 500 μL serum-free medium ± lysenin (100 ng/mL)

    • Incubate at 37°C for 2h

    • Replace with 250 μL fresh serum-free medium

    • Add 250 μL MTT solution (5 mg/mL in PBS)

    • Incubate at 37°C for 3h

    • Measure cell viability

• WNT signaling activity: Measure changes in β-catenin-dependent transcription and LRP6 phosphorylation status

How does CSNK1G3 regulate oxidative stress response via the NADPH dual oxidase complex?

Recent research has revealed CSNK1G3's role in regulating oxidative stress response through interaction with the NADPH dual oxidase complex:

• Physical interaction mechanism:

  • CSNK1G3 physically interacts with dual oxidase maturation factor (DOXA-1)

  • This interaction has been demonstrated through immunoprecipitation and pull-down assays:

    • BL21 bacteria expressing pCold TF::csnk-1_cDNA and pCold TF::doxa-1_cDNA were induced with IPTG

    • Cell lysates were incubated with His-tag resin

    • HEK293T cells were transfected with FLAG-tagged CSNK1G3 or HA-tagged DOXA-1

    • Pull-down assays confirmed physical interaction between the proteins

• Functional consequences:

  • CSNK1G3 promotes ROS levels through regulation of the NADPH dual oxidase complex

  • Loss of CSNK1G3 confers resistance to oxidative stressors

  • This function appears conserved from C. elegans (CSNK-1) to mammals

• Experimental approaches to study this interaction:

  • Co-immunoprecipitation using anti-HA or anti-FLAG antibodies

  • Western blotting with appropriate antibodies

  • Measurement of ROS levels in CSNK1G3-manipulated cells

  • Oxidative stress challenge assays (e.g., iodide excess)

What is the significance of CSNK1G3's proximity protein network for functional studies?

Understanding CSNK1G3's protein interaction network provides critical insights into its cellular functions:

• Proximity biotinylation approach:

  • miniTurbo (mT) biotin ligase fused to CSNK1G3 has been used to map proximal proteins

  • Advantages over BirA* include shorter biotin labeling window

  • This technique biotinylates surface-exposed lysine residues on proximal proteins, enabling their identification by mass spectrometry

• Key identified proximity interactions:

  • β-catenin, ZDHHC8, and PIK3R3 were the most abundant proximal proteins

  • WNT signaling components: DVL, APC, LRP6, FZD3

  • Planar cell polarity proteins: CELSR2, VANGL1

• Differential interactions between family members:

  • Of 548 high-confidence protein interactions, 326 were common to all CK1G subfamily members

  • LRP6 and FZD3 were identified as CSNK1G3-specific proximal proteins

  • Majority of prey proteins localize to the plasma membrane

• Functional significance:

  • CSNK1G3 uniquely activates β-catenin-dependent WNT signaling

  • Its proximity to LRP6 correlates with its ability to induce LRP6 phosphorylation

  • Potential for discovering novel therapeutic targets, as CSNK1G3 is considered a "dark kinase"

How do selective inhibitors of CSNK1G3 affect WNT signaling pathways?

Understanding how CSNK1G3 inhibitors impact WNT signaling has important implications for research and potential therapeutic applications:

• Inhibitor characteristics:

  • Two moderately selective and potent small-molecule inhibitors of the CSNK1G family have been characterized

  • These inhibitors show specificity for the CSNK1G family over other casein kinases

• Effects on WNT signaling:

  • Chemical inhibition of CSNK1G family suppresses WNT signaling

  • Suppression of CSNK1G3 alone had no effect, suggesting functional redundancy within the family

  • Inhibitors suppress, but do not eliminate, WNT-driven LRP6 phosphorylation and β-catenin stabilization

• Comparison with genetic approaches:

  • siRNA-mediated silencing of CSNK1G3 alone had no impact on WNT signaling

  • Co-silencing of all three family members decreased WNT pathway activity

  • CSNK1G3 kinase-dead mutant suppressed WNT signaling

• Experimental design considerations:

  • Use inhibitors as tool compounds to probe CSNK1G family function

  • Compare chemical inhibition with genetic approaches for comprehensive understanding

  • Consider potential compensatory mechanisms within the CSNK1G family when targeting individual members

What are the optimal storage and handling conditions for CSNK1G3 Antibody, Biotin conjugated?

To maintain antibody efficacy and extend shelf life, follow these research-validated storage and handling procedures:

When preparing working dilutions, use buffers without sodium azide if downstream applications involve HRP detection systems, as azide inhibits peroxidase activity.

How can I optimize the signal-to-noise ratio when using CSNK1G3 Antibody, Biotin conjugated in immunoassays?

To achieve optimal signal specificity and minimize background when using biotin-conjugated antibodies:

• Block endogenous biotin:

  • Pre-incubate samples with avidin followed by biotin (avidin-biotin blocking kit)

  • Particularly important for tissues with high endogenous biotin (liver, kidney, brain)

• Optimize antibody concentration:

  • Perform titration experiments to determine optimal concentration

  • ELISA: Start with recommended 1:20,000-1:40,000 dilution

  • IHC: Begin with 1:200 dilution and adjust as needed

• Reduce non-specific binding:

  • Use blocking buffers containing 1-5% BSA or serum from the species of secondary reagent

  • Include 0.1-0.3% Triton X-100 for membrane permeabilization if needed

  • Add 0.05-0.1% Tween-20 to wash buffers

• Detection system optimization:

  • Use streptavidin-HRP at 0.1-1.0 μg/ml

  • For fluorescent detection, try streptavidin-Alexa Fluor conjugates

  • Consider signal amplification systems like TSA (tyramide signal amplification) for low-abundance targets

• Background reduction strategies:

  • If high background persists, try reducing primary antibody concentration

  • Increase washing duration and number of washes

  • Use neutral detergents in wash buffers (0.05% Tween-20)

  • Pre-absorb antibody with relevant tissue lysates

What approaches can resolve data inconsistencies when studying CSNK1G3 in different experimental models?

When faced with conflicting data across different experimental systems, consider these methodological approaches:

• Cell type-specific differences:

  • CSNK1G3 functions vary between cell types – HEK293T cells show different responses than LRP5/6 knockout cells

  • Solution: Include multiple cell lines in analysis and explicitly state the cellular context when reporting results

• Family member redundancy:

  • Individual knockdown of CSNK1G3 shows minimal effects, while triple knockdown of all family members produces strong phenotypes

  • Solution: Design experiments that account for potential compensatory mechanisms among family members

• Isoform-specific effects:

  • CSNK1G3 contains a unique 33 amino acid insertion in its C-terminal domain

  • Solution: Use domain-specific antibodies or constructs to distinguish specific isoform activities

• Post-translational modifications:

  • Phosphorylation status affects function (e.g., CSNK1G1/2/3 phospho-Tyr263)

  • Solution: Include phospho-specific antibodies in your analysis, like the Immunotag™ Casein Kinase Iγ1/2/3 (phospho Tyr263) Polyclonal Antibody

• Experimental protocol variations:

  • Differences in WNT3A stimulation timing can affect results

  • Solution: Standardize protocols across experiments and clearly document all experimental conditions

A systematic approach to resolving inconsistencies includes:

• Validation across multiple experimental systems

• Use of both gain-of-function and loss-of-function approaches

• Correlation of biochemical data with functional outcomes

• Consideration of post-translational modifications and protein interactions

How might CSNK1G3-specific antibody conjugates be leveraged for targeted therapeutic delivery?

Recent advances in antibody-drug conjugates (ADCs) and antibody-oligonucleotide conjugates (AOCs) suggest potential applications for CSNK1G3 antibodies:

• Therapeutic potential based on CSNK1G3 biology:

  • CSNK1G3's unique role in WNT signaling makes it a potential target in cancer contexts where WNT is dysregulated

  • Its involvement in oxidative stress response pathways suggests applications in conditions with ROS dysregulation

• Antibody conjugation approaches:

  • Biotin-streptavidin linkage provides a versatile platform for secondary conjugation

  • Recent advances in AOC technology demonstrate the potential for antibody-mediated delivery of therapeutic oligonucleotides

  • Conjugation methods include:

    • Direct chemical conjugation

    • Avidin-based conjugation

    • Click chemistry approaches (DBCO-azide)

• Delivery considerations:

  • Cell-type specificity based on CSNK1G3 expression patterns

  • Subcellular localization targeting (CSNK1G3 is found at the plasma membrane)

  • Payload selection based on desired mechanism (siRNA, cytotoxic agents)

• Technical challenges:

  • Ensuring antibody specificity for CSNK1G3 over other family members

  • Optimizing drug-to-antibody ratio

  • Maintaining stability of the conjugate in vivo

What are the implications of CSNK1G3's role in oxidative stress response for neurodegenerative disease research?

Emerging research on CSNK1G3's role in oxidative stress regulation suggests potential implications for neurodegenerative diseases:

• Mechanistic relevance:

  • Oxidative stress is a key pathological feature in neurodegenerative diseases including Alzheimer's, Parkinson's, and ALS

  • CSNK1G3 regulates ROS levels through interaction with the NADPH dual oxidase complex

  • WNT signaling dysregulation is implicated in neurodegeneration, and CSNK1G3 has a unique role in this pathway

• Research approaches:

  • Investigate CSNK1G3 expression and activity in neurodegenerative disease models

  • Examine genetic associations between CSNK1G3 variants and disease risk

  • Evaluate effects of CSNK1G3 modulation on neuronal oxidative stress

  • Explore potential neuroprotective effects of CSNK1G3 inhibition or activation

• Experimental models:

  • Neuronal cell lines with CSNK1G3 manipulation

  • Patient-derived iPSCs differentiated into relevant neural cell types

  • Animal models with CSNK1G3 knockout or overexpression in specific brain regions

• Potential therapeutic implications:

  • CSNK1G3 inhibitors as neuroprotective agents

  • Targeted delivery of CSNK1G3 modulators to affected brain regions

  • Biomarkers of CSNK1G3 activity as diagnostic or prognostic indicators