CSNK1D Antibody

What is CSNK1D and what biological functions does it regulate?

CSNK1D is an essential serine/threonine-protein kinase that regulates diverse cellular growth and survival processes. Its primary functions include:

• Regulation of Wnt signaling pathway

• Maintenance of circadian rhythms

• Involvement in DNA repair mechanisms

• Cell proliferation and differentiation control

Also known as CKI-delta, CKId, or Tau-protein kinase CSNK1D, this protein has a predicted molecular weight of approximately 47 kDa . Recent studies have highlighted its significance in cancer progression, particularly in hepatocellular carcinoma (HCC), where it influences tumor proliferation and metastasis .

What applications are CSNK1D antibodies validated for?

CSNK1D antibodies have been validated for multiple experimental applications:

• Western Blotting (WB): For detecting CSNK1D protein expression levels in cell or tissue lysates

• Immunoprecipitation (IP): To isolate CSNK1D and its binding partners

• Immunohistochemistry (IHC): For visualizing CSNK1D expression in tissue sections, particularly in paraffin-embedded samples

• Immunofluorescence (IF): To determine subcellular localization of CSNK1D

• ELISA: For quantitative measurement of CSNK1D levels

Each antibody may have specific optimal applications. For example, ab264278 (rabbit polyclonal) has been validated for IP and WB applications with human samples , while other antibodies like ABIN391599 are validated for WB and IHC with paraffin-embedded sections .

What are the optimal conditions for Western blotting with CSNK1D antibodies?

Based on published protocols, the following conditions are recommended for Western blotting:

• Antibody concentration: 0.04-1 μg/mL depending on the specific antibody and sample type

• Sample loading: 5-50 μg of whole cell lysate (titration recommended for optimal results)

• Expected band size: 47 kDa

• Exposure time: Typically 30 seconds to 3 minutes

For example, ab264278 has been successfully used at 0.04 μg/mL concentration with HeLa whole cell lysate loaded at 5, 15, and 50 μg, using ECL detection technique with 30 seconds exposure time . Different antibodies may require optimization of these parameters based on their specific properties and the experimental system.

How should CSNK1D antibodies be validated before experimental use?

Proper validation is critical for ensuring reliable experimental results. Recommended validation approaches include:

• Positive and negative controls:

  • Positive controls: Cell lines with known CSNK1D expression (e.g., HeLa cells)

  • Negative controls: CSNK1D-knockdown cells or CSNK1D-knockout models

• Multiple detection methods:

  • Verifying consistent results across different techniques (WB, IHC, IF)

  • Confirming subcellular localization patterns match known CSNK1D distribution

• Specificity testing:

  • Peptide competition assay to confirm epitope specificity

  • Comparison with multiple antibodies targeting different CSNK1D regions

For immunoprecipitation validation, comparing results between the target antibody and control IgG is essential, as demonstrated in validation studies with ab264278 .

What species reactivity is available for CSNK1D antibodies?

CSNK1D antibodies are available with various species reactivity profiles:

When selecting an antibody, researchers should prioritize those validated for their specific species of interest and experimental application.

How does CSNK1D expression correlate with cancer prognosis?

CSNK1D has emerged as a significant prognostic marker in multiple cancer types:

Kaplan-Meier analyses from TCGA data have confirmed these correlations, establishing CSNK1D as an independent prognostic biomarker, particularly in hepatocellular carcinoma. Statistical analyses using both TCGA and ICGC datasets support the robustness of CSNK1D as a prognostic indicator .

What is the relationship between CSNK1D and the Wnt/β-catenin signaling pathway?

CSNK1D plays a critical role in regulating the Wnt/β-catenin pathway, particularly in cancer:

• CSNK1D contributes to HCC progression by activating the Wnt/β-catenin signaling pathway

• Mechanistically, CSNK1D interacts with Dishevelled Segment Polarity Protein 3 (DVL3) to mediate this activation

• Silencing CSNK1D significantly reduces Wnt/β-catenin pathway activity, while overexpression enhances it

• Immunofluorescence studies have demonstrated co-localization of CSNK1D with β-catenin and DVL3 using specific antibodies diluted at 1:50 (β-catenin #8480, CST; CSNK1D #sc-55553, SantaCruz; DVL3 #13,444–1-AP, Proteintech)

This relationship suggests CSNK1D could be a therapeutic target for inhibiting oncogenic Wnt/β-catenin signaling in cancer cells.

How can CSNK1D expression be experimentally manipulated to study its functions?

Several approaches have been successfully employed to modulate CSNK1D expression:

Knockdown strategies:

• ShRNA targeting CSNK1D: The sequence CUAUCUCGGUACGGACAUUTTAAUGUCCGUACCGAGAUAGTT has shown high intervening efficacy

• siRNA-mediated knockdown for transient expression reduction

Overexpression approaches:

• Plasmids encoding CSNK1D (e.g., pTSB-CMV-CSNK1D-copGFP-F2A-PuroR)

• Transfection using Lipofectamine 3000, followed by puromycin selection (5 μg/ml) for 14 days to establish stable cell lines

Functional validation:

• CCK-8, EdU, wound healing, and Transwell assays can assess the impact of CSNK1D manipulation on cell proliferation, migration, and invasion

• Western blotting with CSNK1D antibodies (1:200 dilution) to confirm expression changes

These tools allow comprehensive functional analysis of CSNK1D in various cellular contexts.

What is the correlation between CSNK1D expression and immune cell infiltration in tumors?

Recent research has revealed significant associations between CSNK1D and the tumor immune microenvironment:

• CSNK1D expression negatively correlates with immune scores in multiple cancers including BRCA, THYM, THCA, CESC, KIPAN, LUSC, STES, KIRP, HNSC, LUAD, SKCM, UCEC, BLCA, and SARC

• Positive correlation with immune scores was observed in DLBC and LAML

Regarding specific immune cell populations:

• High CSNK1D levels positively associate with CD4+ T cells, neutrophils, and NK cells across most cancer types

• In hepatocellular carcinoma (LIHC), CSNK1D shows strong positive correlations with monocytic lineage cells, M1 and M2 macrophages, and dendritic cells

These findings suggest CSNK1D may serve as a biomarker for immunotherapy response prediction and provide insights into tumor-immune interactions.

How can CSNK1D antibodies be used to investigate protein-protein interactions?

CSNK1D antibodies enable several approaches for studying protein interactions:

Immunoprecipitation (IP):

• Successfully demonstrated with antibodies like ab264278 at 10 μg/mg lysate concentration

• Can capture CSNK1D interacting proteins when followed by mass spectrometry or western blotting

Co-immunoprecipitation (Co-IP):

• Effective for confirming direct interactions, such as CSNK1D-DVL3 binding

• Requires optimization of antibody concentration and buffer conditions

Immunofluorescence co-localization:

• Using CSNK1D antibodies (1:50-1:100 dilution) with antibodies against potential interaction partners

• Visualization with appropriate fluorescent secondary antibodies:

  • Red rabbit/mouse fluorescent antibody (1:100, #AS007/#AS008, ABclonal)

  • Green fluorescent antibody (1:100, #AS011, ABclonal)

  • Nuclear visualization with DAPI

These approaches have successfully demonstrated CSNK1D's interactions with components of the Wnt signaling pathway and could be applied to identify novel interaction partners.

What are the optimal fixation and permeabilization conditions for immunofluorescence with CSNK1D antibodies?

For successful immunofluorescence detection of CSNK1D:

• Fixation: 4% formaldehyde is recommended for cell preservation

• Permeabilization: 0.25% Triton X-100 allows antibody access to intracellular CSNK1D

• Blocking: 1% BSA for 1 hour at room temperature minimizes non-specific binding

• Primary antibody incubation: 12 hours with optimized dilution (typically 1:50-1:100)

• Secondary antibody: Fluor-labeled antibodies specific to the host species of primary antibody

These conditions have been validated for detecting CSNK1D and its co-localization with interaction partners like β-catenin and DVL3 in various cell types.

How can researchers troubleshoot non-specific binding with CSNK1D antibodies?

When encountering non-specific binding issues:

• Antibody dilution optimization:

  • Test a range of dilutions to find the optimal signal-to-noise ratio

  • Start with manufacturer's recommended range and adjust as needed

• Blocking optimization:

  • Increase blocking time or concentration (e.g., 3-5% BSA or serum)

  • Use blocking agent that matches the host species of the secondary antibody

• Validation controls:

  • Include CSNK1D knockdown samples as negative controls

  • Perform peptide competition assays to confirm specificity

• Cross-reactivity assessment:

  • Choose antibodies with minimal cross-reactivity to related kinases

  • Validate specificity in systems with known CSNK1D expression patterns

• Sample preparation improvements:

  • Ensure complete cell lysis for Western blotting

  • Optimize fixation time for immunohistochemistry and immunofluorescence

How do CSNK1D expression patterns differ across cancer types and what are the implications for antibody selection?

CSNK1D expression varies significantly across cancer types, impacting antibody selection strategies:

When studying CSNK1D in specific cancer contexts:

• Select antibodies validated in the relevant cancer type

• Consider epitope accessibility, which may be affected by cancer-specific post-translational modifications

• For cancer tissue microarrays, antibodies validated for IHC in paraffin-embedded sections are essential

These considerations ensure optimal detection sensitivity and specificity across different cancer models.

How might CSNK1D antibodies contribute to therapeutic development in cancer?

CSNK1D antibodies can facilitate therapeutic development through several approaches:

• Target validation: Confirming CSNK1D's role in cancer progression through immunohistochemical analysis of patient samples

• Mechanism elucidation: Identifying CSNK1D-dependent signaling pathways using antibody-based techniques

• Biomarker development: Using CSNK1D antibodies to stratify patients for clinical trials

• Therapeutic monitoring: Assessing CSNK1D inhibition in response to experimental treatments

Given CSNK1D's established role in HCC progression and sorafenib resistance, antibody-based studies could particularly advance therapies for liver cancer and other malignancies where CSNK1D serves as a negative prognostic factor .

What are the key considerations for using CSNK1D antibodies in multiplex immunofluorescence studies?

When designing multiplex immunofluorescence experiments:

• Antibody compatibility:

  • Select CSNK1D antibodies from different host species than other target antibodies

  • Ensure secondary antibodies lack cross-reactivity

• Signal optimization:

  • Balance fluorophore brightness to avoid channel bleed-through

  • Optimize antibody concentrations for each target individually before multiplexing

• Controls for co-localization studies:

  • Include single-stained controls for each antibody

  • Use appropriate negative controls for each target protein

• Sequential staining protocols:

  • Consider tyramide signal amplification for sequential staining if using antibodies from the same host species

  • Validate complete stripping between rounds if performing sequential staining

These approaches enable sophisticated analysis of CSNK1D's relationships with other proteins in complex cellular contexts.

How should researchers design experiments to study CSNK1D's role in circadian rhythm regulation?

When investigating CSNK1D's circadian functions:

• Cell synchronization protocols:

  • Synchronize cells using serum shock or dexamethasone treatment

  • Collect samples at regular intervals across 24-48 hours

• Detection methods:

  • Western blotting with CSNK1D antibodies to assess protein levels

  • Immunofluorescence to track subcellular localization changes throughout the circadian cycle

• Functional assessments:

  • Compare CSNK1D knockdown/overexpression effects on clock gene expression

  • Analyze period length and amplitude changes in circadian reporter assays

• Interaction studies:

  • Use co-IP with CSNK1D antibodies to detect time-dependent interactions with clock proteins

  • Perform kinase assays to measure CSNK1D activity toward circadian substrates

These approaches can reveal how CSNK1D dynamically regulates circadian mechanisms, complementing its known roles in cancer progression .