CSNK1G3 Antibody

What is CSNK1G3 and what are its key biological functions?

CSNK1G3 (Casein Kinase 1, gamma 3) is a member of the casein kinase 1 family that regulates various cellular functions. One of its critical roles involves the regulation of ceramide transport protein (CERT), which delivers ceramide to the Golgi apparatus for sphingomyelin synthesis. CSNK1G3 inactivates CERT through multiple phosphorylation events, thus controlling sphingomyelin production . The protein has a molecular weight of approximately 51 kDa and contains a kinase domain and a regulatory C-terminal region that is crucial for its proper subcellular localization and function .

Recent studies have also identified a circular RNA form (circCSNK1G3) that is upregulated in renal cell carcinoma and may promote cancer progression . The functional significance of CSNK1G3 is further highlighted by its distribution in post-Golgi compartments, including late endosomes, recycling endosomes, and lysosomes, as well as distal Golgi compartments .

How should CSNK1G3 antibodies be optimized for different experimental applications?

Optimization of CSNK1G3 antibodies requires systematic titration for each application:

For Western Blotting:

• Begin with dilutions of 1:100-1:500 for most commercial CSNK1G3 antibodies

• Use appropriate blocking agents (5% BSA or non-fat milk) to reduce non-specific binding

• Include proper controls (CSNK1G3 knockout cells or tissues) to confirm band specificity

• Expected molecular weight: ~51 kDa

For Immunohistochemistry:

• Use 1:50-1:100 dilutions for paraffin-embedded sections

• Optimize antigen retrieval conditions (typically citrate buffer pH 6.0 or EDTA pH 8.0)

• Validate specificity with positive and negative tissue controls

For ELISA:

• Start with 1:1000 dilution and adjust based on signal strength

• Titrate primary and secondary antibody concentrations independently

• Include standard curves using recombinant CSNK1G3 protein when possible

Optimization should be performed for each new lot of antibody and for each specific cell line or tissue type being studied.

What are the most effective methods for validating CSNK1G3 antibody specificity?

Comprehensive validation of CSNK1G3 antibodies should include:

• Genetic validation: Compare antibody reactivity between wild-type cells and CSNK1G3 knockout cells generated through CRISPR/Cas9 genome editing . This represents the gold standard for antibody validation.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to the sample. Specific signals should be significantly reduced or eliminated.

• Molecular weight verification: Confirm that the detected band matches the expected molecular weight of CSNK1G3 (51 kDa) .

• Multiple antibody approach: Use antibodies targeting different epitopes of CSNK1G3 (N-terminal and C-terminal) to confirm consistent results .

• siRNA knockdown: Transiently reduce CSNK1G3 expression and confirm corresponding reduction in antibody signal.

• Functional validation: For C-terminal targeting antibodies, verify using the lysenin-resistance assay described in the literature, which can identify the functional consequences of C-terminal truncation .

How does C-terminal truncation of CSNK1G3 affect its function and localization?

C-terminal truncation of CSNK1G3 has profound effects on both its cellular localization and function:

Localization changes:

• Wild-type CSNK1G3 is primarily distributed to post-Golgi compartments (late endosomes, recycling endosomes, lysosomes) and less frequently to distal Golgi compartments

• C-terminally truncated CSNK1G3 (either CΔ20 or CΔ38) relocates to the cytosol and nucleus, losing its association with punctate compartments

• This indicates that the C-terminal region contains determinants essential for proper subcellular targeting

Functional consequences:

• Truncation of the C-terminal region (as little as 20 amino acids) causes down-regulation of sphingomyelin synthesis

• Cells expressing C-terminally truncated CSNK1G3 display resistance to lysenin, a sphingomyelin-binding cytolysin

• The lysenin-resistance phenotype provides a functional readout for successful genome editing of the CSNK1G3 C-terminus

These findings suggest that the C-terminal region plays a critical role in regulating CSNK1G3's activity, likely by influencing its substrate interactions and cellular compartmentalization.

What methods can be used to study the interaction between CSNK1G3 and its substrates?

Several complementary approaches can be employed to investigate CSNK1G3-substrate interactions:

• LanthaScreen® Eu Kinase Binding Assay:

  • This assay measures binding of fluorescent tracers to CSNK1G3 and competition by inhibitors

  • Allows determination of inhibitor binding constants (Kd values) and IC50 values

  • Useful for screening potential small molecule modulators of CSNK1G3 activity

• Co-immunoprecipitation:

  • Use anti-CSNK1G3 antibodies to pull down the kinase and associated substrate proteins

  • Western blot analysis with substrate-specific antibodies can confirm interactions

  • Can be performed in native conditions to preserve physiological interactions

• Kinase assays:

  • In vitro phosphorylation assays using purified CSNK1G3 and candidate substrates

  • Detection of phosphorylation using phospho-specific antibodies or radioactive ATP

• Proximity ligation assay (PLA):

  • Detects protein interactions in fixed cells with single-molecule resolution

  • Particularly useful for detecting transient kinase-substrate interactions

  • Compatible with CSNK1G3 antibodies validated for immunocytochemistry

• Biotin-coupled RNA pull-down assay:

  • Specifically used for studying interactions between circCSNK1G3 and miRNAs

  • Relies on 3'-end biotinylated miRNA mimics or circCSNK1G3

  • Streptavidin-coated magnetic beads are used to pull down the RNA complexes

What is known about the role of circCSNK1G3 in cancer research?

Recent research has identified a circular RNA form of CSNK1G3 (circCSNK1G3) with significant implications for cancer biology:

Expression patterns:

• circCSNK1G3 shows high expression in renal cell carcinoma (RCC) cell lines compared to normal human renal cell lines

• Higher expression of CSNK1G3 (both linear mRNA and circular RNA) correlates with worse prognosis in RCC patients according to TCGA-KICH database analysis

Molecular characteristics:

• Unlike linear CSNK1G3 mRNA, circCSNK1G3 lacks a poly-A tail and is resistant to RNase R digestion

• circCSNK1G3 exhibits greater stability and a longer half-life compared to linear CSNK1G3 mRNA

• Fluorescence in situ hybridization (FISH) demonstrates that circCSNK1G3 is predominantly distributed in the cytoplasm

Functional role:

• circCSNK1G3 appears to up-regulate miR-181b, which may promote growth in RCC

• The interaction between circCSNK1G3 and miR-181b can be studied using biotin-coupled RNA pull-down assays and luciferase reporter assays

This emerging research suggests circCSNK1G3 may serve as a potential biomarker or therapeutic target in RCC and possibly other cancers.

How can researchers overcome common challenges in CSNK1G3 detection by Western blotting?

When encountering difficulties with CSNK1G3 detection in Western blotting, consider the following methodological adjustments:

Problem: Weak or absent signal

• Solution: Increase antibody concentration (try 1:100 instead of 1:500)

• Solution: Extend primary antibody incubation time (overnight at 4°C)

• Solution: Enhance signal using more sensitive detection systems (chemiluminescent substrates with longer emission times)

• Solution: Increase protein loading (50-100 μg of total protein)

Problem: Multiple bands or non-specific binding

• Solution: Optimize blocking conditions (try 5% BSA instead of milk)

• Solution: Increase wash stringency (use 0.1% Tween-20 in TBS and extend wash times)

• Solution: Pre-absorb the antibody with cell lysates from CSNK1G3 knockout cells

• Solution: Use freshly prepared lysates with complete protease inhibitor cocktails

Problem: Detecting C-terminally truncated CSNK1G3

• Solution: Use antibodies targeting the N-terminal region

• Solution: Consider generating custom antibodies against the kinase domain

• Solution: Compare migration patterns with recombinant CSNK1G3 constructs of known truncations

Problem: Distinguishing CSNK1G3 from other CK1 family members

• Solution: Include positive controls expressing only CSNK1G3

• Solution: Run parallel blots with antibodies specific for other CK1 isoforms

• Solution: Use high-resolution SDS-PAGE (10-12% gels) to achieve better separation

What approaches can improve CSNK1G3 antibody performance in immunohistochemistry?

For optimal immunohistochemical detection of CSNK1G3, consider these methodological refinements:

Antigen retrieval optimization:

• Test multiple antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0, EDTA buffer pH 8.0, or Tris-EDTA pH 9.0)

• Optimize retrieval time (10-30 minutes) and method (microwave, pressure cooker, or water bath)

Signal amplification strategies:

• Implement tyramide signal amplification systems for low-abundance targets

• Use polymer-based detection systems instead of traditional ABC methods

• Consider sequential antibody application for dual staining with other markers

Background reduction:

• Pre-incubate sections with hydrogen peroxide (3% for 10 minutes) to block endogenous peroxidase

• Use avidin-biotin blocking for biotin-based detection systems

• Include species-specific normal serum (2-5%) in blocking solution

Antibody optimization:

• Dilute antibodies in buffers containing 0.05-0.1% non-ionic detergents

• Extend primary antibody incubation time (overnight at 4°C)

• For C-terminal antibodies, validate with tissues from the CSNK1G3ΔC model to confirm specificity

Validation controls:

• Include tissue sections known to express CSNK1G3 (positive control)

• Process serial sections with isotype control antibodies at the same concentration

• Compare staining patterns with multiple antibodies targeting different CSNK1G3 epitopes

What are promising future applications for CSNK1G3 antibodies in research?

As research continues to uncover the diverse functions of CSNK1G3, several promising applications for CSNK1G3 antibodies are emerging:

Single-cell analysis:

• Adaptation of CSNK1G3 antibodies for mass cytometry (CyTOF)

• Integration with single-cell RNA sequencing to correlate protein levels with transcriptomic data

• Development of phospho-specific antibodies to monitor CSNK1G3 activation at the single-cell level

Therapeutic monitoring:

• Development of antibodies specific to circCSNK1G3 for monitoring cancer progression

• Creation of companion diagnostics for potential CSNK1G3 inhibitors in clinical trials

• Monitoring CSNK1G3 compartmentalization as a biomarker for cellular stress responses

Advanced imaging applications:

• Super-resolution microscopy to visualize CSNK1G3 subcellular distribution with nanometer precision

• Live-cell imaging using nanobody derivatives of validated CSNK1G3 antibodies

• Multiplexed imaging to study CSNK1G3 interactions with substrates in native contexts

Therapeutic antibody development:

• Engineering of antibodies that can modulate CSNK1G3 activity in specific subcellular compartments

• Development of antibody-drug conjugates targeting cancer cells with abnormal CSNK1G3 expression

• Intrabody applications to manipulate CSNK1G3 function in specific cellular compartments

These emerging applications highlight the continued importance of developing and characterizing specific, well-validated CSNK1G3 antibodies for both basic research and potential clinical applications.