CSNK1G3 Antibody, HRP conjugated

What is the optimal dilution range for CSNK1G3 antibody, HRP conjugated in ELISA applications?

For ELISA applications, the CSNK1G3 antibody, HRP conjugated, should be used at a dilution range of 1:20,000-1:40,000. This recommendation is based on validated protocols for detecting endogenous levels of CSNK1G3 protein . For optimal results, researchers should perform preliminary titration experiments with positive and negative controls to determine the ideal antibody concentration for their specific experimental system.

How should CSNK1G3 antibody, HRP conjugated be stored to maintain optimal activity?

The CSNK1G3 antibody, HRP conjugated should be stored at -20°C for long-term storage (up to one year from the date of receipt) . For frequent use and short-term storage (up to one month), the antibody can be kept at 4°C to avoid repeated freeze-thaw cycles that may compromise antibody performance . The antibody is typically supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . Always aliquot the antibody upon first thaw to minimize freeze-thaw cycles, and centrifuge briefly before use to collect contents at the bottom of the tube.

What is the species reactivity profile of commercially available CSNK1G3 antibodies?

Commercial CSNK1G3 antibodies show various reactivity profiles depending on the manufacturer and clone. Based on available data, most CSNK1G3 antibodies demonstrate reactivity with human, mouse, and rat samples . Some antibodies offer expanded cross-reactivity to additional species:

When selecting an antibody for cross-species applications, validation of reactivity in the target species is recommended through preliminary experiments.

How can I optimize detection of CSNK1G3 in Western blotting applications using HRP-conjugated antibodies?

For optimal detection of CSNK1G3 in Western blotting using HRP-conjugated antibodies, follow this methodological approach:

• Sample preparation: Prepare cell lysates using a lysis buffer containing protease inhibitors. For CSNK1G3 detection, cells such as HeLa, sp2/0, and PC12 have been validated as positive controls .

• Protein loading and separation: Load 20-40 μg of total protein per lane. CSNK1G3 has a calculated molecular weight of approximately 51 kDa . Use 10-12% SDS-PAGE gels for optimal separation.

• Transfer conditions: Transfer proteins to PVDF membrane (preferred over nitrocellulose for CSNK1G3 detection) at 100V for 60-90 minutes in cold transfer buffer containing 20% methanol.

• Blocking: Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Antibody incubation: Dilute HRP-conjugated CSNK1G3 antibody to 1:1,000-1:3,000 in blocking buffer and incubate overnight at 4°C .

• Washing and detection: Wash membranes 3-5 times with TBST. Directly proceed to chemiluminescent detection since the antibody is already HRP-conjugated.

• Expected results: CSNK1G3 should be detected at approximately 51 kDa. Validation experiments have confirmed this band in multiple cell lines including HeLa, sp2/0, and PC12 cells .

For troubleshooting weak signals, consider extending exposure time, increasing antibody concentration, or using enhanced chemiluminescent substrates specifically designed for HRP detection.

What are the methodological considerations for detecting CSNK1G3 interactions with RIP3 using co-immunoprecipitation approaches?

When investigating CSNK1G3 interactions with RIP3 through co-immunoprecipitation experiments, consider the following methodological approach based on published research:

• Cell system selection: HeLa cells expressing HA-3×Flag-RIP3 have been successfully used for investigating casein kinase interactions with RIP3 . For CSNK1G3 specifically, transfection with Myc-tagged CSNK1G3 allows for detection of the interaction.

• Experimental design:

  • Transfect cells with expression vectors for tagged versions of both proteins

  • Include proper controls: vector-only control, kinase-dead mutants (K75A for casein kinases), and single protein expressions

  • Consider including necroptosis stimuli (TSZ: TNF-α, Smac mimetic, and z-VAD-fmk) to assess interaction under stimulated conditions

• Co-IP procedure:

  • Prepare cell extracts using a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and protease/phosphatase inhibitors

  • Perform immunoprecipitation using anti-tag antibodies (anti-Flag or anti-Myc)

  • Wash immunoprecipitates at least three times with lysis buffer

  • Elute bound proteins by either competitive elution with antigenic peptide (0.5 mg/mL) or by boiling in SDS loading buffer

• Detection strategy:

  • Western blot analysis using antibodies against both proteins

  • For CSNK1G3-RIP3 interactions, examine both the co-IP of CSNK1G3 with RIP3 and the effect on RIP3 phosphorylation (Ser227)

  • Assess downstream effects on RIP1-RIP3 interaction and MLKL phosphorylation

Note that while CSNK1G2 has been documented to interact with and inhibit RIP3 activation, the specific role of CSNK1G3 in this pathway requires further investigation, as CSNK1G3 was not found to suppress RIP3 kinase activity as effectively as CSNK1G2 in published studies .

What are the technical considerations for CSNK1G3 gene knockout experiments using CRISPR-Cas9?

For CRISPR-Cas9 mediated knockout of CSNK1G3, researchers should follow this methodological framework:

• sgRNA design and vector preparation:

  • Design sgRNAs targeting exonic regions of CSNK1G3 (preferably early exons)

  • According to published protocols, sgRNAs can be cloned into a Cas9 expression plasmid such as pX458-GFP

  • Verify the specificity of sgRNAs using appropriate design tools to minimize off-target effects

• Cell transfection and selection:

  • Transfect target cells (HeLa cells have been validated) with the sgRNA-Cas9 expression plasmid

  • After 24-48 hours, sort GFP-positive cells using FACS to enrich for transfected cells

  • For single-cell cloning, plate sorted cells at low density (0.5-1 cell/well) in 96-well plates

• Verification of genome editing:

  • Screen clones by PCR amplification of the targeted region followed by Sanger sequencing

  • Verify knockout at the protein level via Western blotting using validated CSNK1G3 antibodies

  • Functional verification can include phenotypic assays specific to CSNK1G3 function

• Complementation studies:

  • For rescue experiments, reintroduce wild-type or mutant CSNK1G3 using retroviral vectors (e.g., pMXs-IRES-Neo)

  • Confirm expression by Western blotting using anti-CSNK1G3 or anti-tag antibodies

  • Assess functional rescue through appropriate cellular assays

Key considerations for successful CSNK1G3 knockout include:

• Designing multiple sgRNAs to increase the likelihood of successful editing

• Including proper controls (parental cells, non-targeting sgRNA)

• Thorough validation of knockout at both genomic and protein levels

How can I design experiments to study CSNK1G3's role in Wnt signaling pathways?

To investigate CSNK1G3's role in Wnt signaling, implement the following experimental design:

• Cell system selection:

  • Use cell lines with active Wnt signaling (e.g., HEK293T, L cells, SW480 colorectal cancer cells)

  • Consider CSNK1G3 knockout (via CRISPR-Cas9) and overexpression systems in parallel

• Signaling pathway assessment:

  • Measure canonical Wnt signaling activity using TOPFlash/FOPFlash luciferase reporter assays

  • Assess β-catenin stabilization and nuclear translocation via Western blotting of fractionated cell lysates and immunofluorescence microscopy

  • Examine expression of Wnt target genes (e.g., AXIN2, c-MYC, CCND1) by qRT-PCR

• Rescue experiments:

  • In CSNK1G3 knockout cells, reintroduce wild-type CSNK1G3 or kinase-dead mutants (K75A, D165N) using retroviral vectors

  • Compare with related casein kinase family members (CSNK1G1, CSNK1G2) to assess functional specificity

• Protein interaction analysis:

  • Perform co-immunoprecipitation experiments to identify CSNK1G3 interaction with Wnt pathway components

  • Use proximity ligation assays to confirm interactions in intact cells

  • Consider in vitro kinase assays to identify direct CSNK1G3 substrates within the pathway

• Phosphorylation site mapping:

  • Identify potential CSNK1G3 phosphorylation sites on Wnt pathway components using phospho-specific antibodies

  • Confirm sites using mass spectrometry analysis of immunoprecipitated proteins

This experimental framework allows for comprehensive analysis of CSNK1G3's function within Wnt signaling, from pathway activation to specific molecular mechanisms.

What methodological approaches can be used to compare the functional differences between CSNK1G family members (CSNK1G1, CSNK1G2, and CSNK1G3)?

To systematically investigate functional differences between CSNK1G family members, implement the following methodological approaches:

• Expression vector construction and validation:

  • Clone full-length cDNAs for human CSNK1G1, CSNK1G2, and CSNK1G3 into identical expression vectors with the same epitope tags

  • Confirm expression levels by Western blotting to ensure comparable expression

  • Include kinase-dead mutants (K75A for all three family members) as controls

• Cellular localization comparison:

  • Perform immunofluorescence microscopy using tag-specific antibodies or family member-specific antibodies

  • Generate stable cell lines expressing each family member and compare subcellular localization

  • Analyze potential relocalization under various stimuli (e.g., Wnt pathway activation)

• Substrate specificity assessment:

  • Conduct in vitro kinase assays using purified recombinant CSNK1G proteins

  • Use peptide arrays or proteome-wide approaches to identify specific substrates

  • Verify selected substrates in cellular contexts through phospho-specific antibodies

• Functional redundancy evaluation:

  • Generate single, double, and triple knockout cell lines using CRISPR-Cas9

  • Perform rescue experiments with individual family members in triple knockout backgrounds

  • Assess phenotypic outcomes in pathway-specific assays (e.g., Wnt signaling, necroptosis regulation)

• Protein interaction profiling:

  • Conduct comparative immunoprecipitation followed by mass spectrometry to identify unique and shared interacting partners

  • Compare results with published data showing CSNK1G2 (but not CSNK1G1 or CSNK1G3) interacts with and suppresses RIP3 kinase activity

• Domain swap experiments:

  • Generate chimeric proteins exchanging domains between family members to identify regions responsible for functional specificity

  • Focus on C-terminal domains which often confer substrate specificity in casein kinases

This comprehensive approach will reveal both overlapping and distinct functions of CSNK1G family members, providing insight into their specific roles in various cellular processes.

What strategies can be employed to detect phosphorylated forms of CSNK1G3 in experimental samples?

Detection of phosphorylated CSNK1G3 requires careful methodological considerations:

• Sample preparation optimization:

  • Harvest cells rapidly and lyse in buffer containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • For unstable phosphorylation events, consider crosslinking before lysis or use of phosphatase inhibitor cocktails

  • Maintain samples at 4°C throughout processing

• Enrichment strategies:

  • Perform immunoprecipitation using total CSNK1G3 antibodies to concentrate the protein before phospho-detection

  • Consider phospho-protein enrichment using TiO₂ or immobilized metal affinity chromatography (IMAC)

  • For specific known phosphorylation sites, use phospho-specific antibodies in immunoprecipitation

• Detection methods:

  • Western blotting using phospho-specific antibodies (if available) or general phospho-Ser/Thr antibodies after immunoprecipitation

  • Phos-tag™ SDS-PAGE to separate phosphorylated forms based on mobility shift

  • Mass spectrometry analysis for unbiased identification of phosphorylation sites

• Validation approaches:

  • Lambda phosphatase treatment as a negative control

  • Site-directed mutagenesis of putative phosphorylation sites (convert Ser/Thr to Ala)

  • Kinase inhibitor treatment to prevent specific phosphorylation events

• Analysis of functional significance:

  • Compare phosphorylation patterns after stimuli (e.g., Wnt activation)

  • Assess correlation between phosphorylation state and kinase activity

  • Perform functional assays with phospho-mimetic (Ser/Thr to Asp/Glu) and phospho-resistant (Ser/Thr to Ala) mutants

Based on research on related casein kinases, focus on potential auto-phosphorylation sites and sites phosphorylated by upstream kinases in signaling cascades.

How can I optimize immunohistochemistry protocols for CSNK1G3 detection in tissue samples?

For optimal CSNK1G3 detection in tissue samples via immunohistochemistry, follow these methodological recommendations:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin following standard protocols

  • Cut sections at 4-5 μm thickness onto adhesive slides

• Antigen retrieval optimization:

  • Test both heat-induced epitope retrieval (HIER) methods:
    a) Citrate buffer (pH 6.0): 20 minutes at 95-98°C
    b) EDTA buffer (pH 9.0): 20 minutes at 95-98°C

  • Compare retrieval methods using positive control tissues

• Blocking and antibody incubation:

  • Block endogenous peroxidase activity with 3% hydrogen peroxide

  • Apply protein block (5% normal goat serum)

  • Dilute primary CSNK1G3 antibodies at 1:50-1:200 range

  • Incubate at 4°C overnight for optimal sensitivity

• Detection system:

  • For HRP-conjugated primary antibodies, proceed directly to chromogen development

  • For unconjugated antibodies, use polymer-HRP detection systems

  • Develop with DAB substrate for 5-10 minutes (optimize timing)

  • Counterstain with hematoxylin, dehydrate, and mount

• Controls and validation:

  • Include known positive control tissues

  • Include negative controls (omitting primary antibody)

  • Confirm specificity with pre-absorption using immunizing peptide

  • Compare with validated antibodies using serial sections

• Evaluation criteria:

  • Assess subcellular localization (expected to be primarily cytoplasmic with potential nuclear staining)

  • Evaluate staining intensity (0, 1+, 2+, 3+)

  • Record percentage of positive cells

  • Document cell-type specific expression patterns

The Human Protein Atlas recommends dilutions of 1:20-1:50 for CSNK1G3 antibody HPA027010 specifically for immunohistochemistry applications .

What approaches can be used to study the interaction between CSNK1G3 and necroptosis pathways?

To investigate potential interactions between CSNK1G3 and necroptosis pathways, implement the following research strategy:

• Expression correlation analysis:

  • Compare CSNK1G3 expression with necroptosis markers (RIPK1, RIPK3, MLKL) across cell types

  • Assess changes in CSNK1G3 expression during necroptosis induction

  • Compare with CSNK1G2, which has been shown to inhibit necroptosis by binding to RIPK3

• Protein interaction studies:

  • Perform co-immunoprecipitation experiments between CSNK1G3 and necroptosis components (RIPK1, RIPK3, MLKL)

  • Compare binding efficiency with CSNK1G2, which strongly binds RIPK3

  • Assess whether kinase-dead mutants (CSNK1G3-K75A) can still interact with necroptosis components

• Functional impact assessment:

  • Generate CSNK1G3 knockout and overexpression cell lines

  • Treat with necroptosis inducers (TNF-α + Smac mimetic + z-VAD-fmk, or TSZ)

  • Measure cell death using viability assays (Cell-Titer Glo)

  • Assess RIPK3 phosphorylation status and MLKL phosphorylation

• Kinase activity evaluation:

  • Determine if CSNK1G3 can phosphorylate components of the necroptosis pathway in vitro

  • Compare with CSNK1G2's effect on RIPK3 kinase activity

  • Identify potential phosphorylation sites using mass spectrometry

• In vivo relevance:

  • Analyze tissues from CSNK1G3 knockout mice for altered sensitivity to necroptotic stimuli

  • Compare with published data on CSNK1G2 knockout mice, which showed accelerated TNF-α-induced systematic sepsis

  • Examine CSNK1G3 expression in human samples with necroptosis-associated diseases

This experimental framework will determine whether CSNK1G3 plays a role in necroptosis regulation distinct from or overlapping with CSNK1G2, which has been established as a negative regulator of necroptosis .

How do different fixation and permeabilization methods affect CSNK1G3 antibody binding in immunocytochemistry?

The choice of fixation and permeabilization methods significantly impacts CSNK1G3 antibody binding in immunocytochemistry. Below is a methodological comparison:

• Fixation methods comparison:

• Permeabilization optimization (if using paraformaldehyde fixation):

Empirical testing with your specific CSNK1G3 antibody is recommended as epitope accessibility may vary between different antibody clones and target regions.

What are the methodological considerations for multiplexed detection of CSNK1G3 with other signaling pathway components?

For multiplexed detection of CSNK1G3 with other signaling components, consider the following methodological approaches:

• Antibody selection and validation:

  • Choose antibodies raised in different host species to avoid cross-reactivity

  • Validate individual antibodies separately before multiplexing

  • If using multiple rabbit antibodies, consider sequential detection with HRP conjugates and tyramide signal amplification

  • Test for potential cross-reactivity between detection systems

• Immunofluorescence multiplexing strategies:

• Chromogenic multiplexing for IHC:

  • Use HRP and alkaline phosphatase (AP) conjugates with different substrates

  • Consider sequential chromogenic IHC with antibody stripping between rounds

  • Implement multispectral imaging systems for separation of closely related chromogens

• Mass cytometry approach:

  • For single-cell analysis, consider CyTOF with metal-conjugated antibodies

  • Enables simultaneous detection of >40 parameters without spectral overlap

  • Requires specialized equipment and metal-conjugated antibodies

• Spatial context preservation:

  • For tissue analysis, consider application of multiplex IF with imaging mass cytometry

  • Digital spatial profiling allows multiplexed protein detection with spatial resolution

  • RNAscope combined with protein detection can correlate CSNK1G3 mRNA with protein expression and pathway components

• Protocol optimization for CSNK1G3 with Wnt pathway components:

  • Begin with sequential detection of CSNK1G3 (HRP-conjugated) and β-catenin (using a different detection system)

  • Include phospho-specific antibodies for activated pathway components

  • Incorporate nuclear counterstaining to assess nuclear translocation of pathway components