CSNK2A1 Antibody

What is CSNK2A1 and why is it significant in research?

CSNK2A1 (Casein kinase II subunit alpha) is a serine/threonine kinase that phosphorylates multiple protein substrates and participates in diverse cellular and biological processes including cell cycle progression, apoptosis, transcription, and viral replication. It has been implicated in various human diseases, notably with two missense mutations in the CSNK2A1 gene associated with autism spectrum disorder. The protein is emerging as a promising biomarker and therapeutic target, making reliable antibody detection crucial for advancing research in this field .

What are the molecular characteristics of CSNK2A1 protein?

CSNK2A1 has a calculated molecular weight of 45 kDa, though it typically appears at 40-45 kDa in Western blot analyses . The protein is encoded by the CSNK2A1 gene (GenBank accession number BC011668, NCBI Gene ID 1457) and has the UniProt ID P68400 . As a catalytic subunit of casein kinase 2, it plays important roles in cell cycle control and other signaling pathways, making it detectable in various cellular compartments depending on its functional state .

What experimental applications are CSNK2A1 antibodies validated for?

CSNK2A1 antibodies have been successfully validated for multiple applications including Western blot (WB), immunoprecipitation (IP), immunohistochemistry (IHC), and immunofluorescence/immunocytochemistry (IF/ICC) . Recent standardized validation studies have characterized ten commercial antibodies across these applications using knockout cell line controls, providing researchers with reliable data to select antibodies appropriate for their specific experimental needs .

How do I select the most appropriate CSNK2A1 antibody for my research?

When selecting a CSNK2A1 antibody, consider: 1) The specific application (WB, IP, IF, IHC) you require; 2) Host species compatibility with your experimental system; 3) Clonality (monoclonal vs. polyclonal) based on your needs for specificity vs. epitope coverage; 4) Validation evidence, particularly in knockout systems; and 5) Species reactivity relevant to your research model. Recent characterization studies of ten commercial antibodies provide an excellent resource for making informed selections based on standardized testing protocols .

What cell lines are optimal for CSNK2A1 antibody validation?

Based on transcriptomic analysis from the DepMap database, HAP1 cell lines express CSNK2A1 at levels of 7.0 log₂ (TPM+1), which is above the average range in cancer cells and sufficient for antibody validation experiments . Other cell lines with confirmed CSNK2A1 expression include A431, PC-3, K-562, NIH/3T3, Raji, LNCaP, HeLa, HEK-293, HepG2, Jurkat, MOLT-4, and HL-60 cells . The ideal validation approach involves comparing antibody performance in both wild-type and CSNK2A1 knockout versions of the same cell line, as demonstrated in recent studies .

What validation criteria should be applied to CSNK2A1 antibodies?

The gold standard for antibody validation includes testing in both wild-type and knockout systems. For CSNK2A1, validated antibodies should show: 1) Specific signal at 40-45 kDa in Western blot that is absent in knockout samples; 2) Successful immunoprecipitation of CSNK2A1 from wild-type but not knockout lysates; 3) Distinct immunofluorescence staining patterns in wild-type cells that are absent or significantly reduced in knockout cells; and 4) Reproducible results across multiple experiments. Quantitative analysis of signals in hundreds of wild-type and knockout cells provides statistical confidence in antibody specificity .

What are the optimal protocols for Western blot detection of CSNK2A1?

For Western blot detection of CSNK2A1:

• Cell lysis: Collect cells in RIPA buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with 1× protease inhibitor cocktail

• Process samples: Briefly sonicate lysates and incubate on ice for 30 minutes

• Clarify: Centrifuge at ~110,000×g for 15 minutes at 4°C

• SDS-PAGE: Use equal protein amounts from supernatants

• Antibody dilution: Use at 1:5000-1:50000 dilution (antibody-dependent)

• Expected result: Detection of a 40-45 kDa band that is absent in knockout controls

What methodology should be used for immunofluorescence with CSNK2A1 antibodies?

For optimal immunofluorescence detection of CSNK2A1:

• Cell preparation: Consider a mosaic approach with fluorescently labeled wild-type and knockout cells plated together in optically clear flat-bottom 96-well plates

• Incubation: Culture cells for 24 hours at 37°C with 5% CO₂

• Antibody dilution: Use at 1:50-1:500 or 1:750-1:3000 depending on the specific antibody

• Controls: Include both wild-type and knockout cells in the same field of view to reduce staining and imaging bias

• Analysis: Quantify immunofluorescence intensity across hundreds of cells for statistical confidence

• Expected patterns: Nuclear and cytoplasmic staining that is absent or significantly reduced in knockout cells

How should immunoprecipitation experiments with CSNK2A1 antibodies be designed?

For successful immunoprecipitation of CSNK2A1:

• Antibody amount: Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

• Sample collection: Prepare equal proportions of starting material, unbound fraction, and whole immunoprecipitate eluates

• Analysis: Separate by SDS-PAGE and detect CSNK2A1 using a validated antibody from Western blot experiments

• Controls: Compare results between wild-type and knockout samples to confirm specificity

• Verification: Look for successful capture of CSNK2A1 in the IP fraction from wild-type samples with corresponding depletion in the unbound fraction

How can I optimize antibody dilutions for different CSNK2A1 detection methods?

Regardless of application, it is recommended that each antibody be titrated in your specific experimental system to achieve optimal results .

What are common issues in CSNK2A1 antibody experiments and how can they be addressed?

Common challenges when working with CSNK2A1 antibodies include:

• Cross-reactivity with CSNK2A2 due to sequence homology: Validate using genetic knockout controls specifically for CSNK2A1

• Background signal: Optimize blocking conditions, antibody dilutions, and wash protocols; pre-absorb antibodies with knockout cell lysates if necessary

• Inconsistent results across applications: Some antibodies perform well in certain applications but not others; select application-specific antibodies based on validation data

• Batch-to-batch variability: Include consistent positive and negative controls in each experiment

• Epitope masking: Consider multiple antibodies targeting different regions of CSNK2A1 for comprehensive analysis

How should I interpret contradictory results obtained with different CSNK2A1 antibodies?

When faced with contradictory results:

• Prioritize data from antibodies validated in genetic knockout systems

• Consider epitope accessibility issues that might affect certain antibodies

• Evaluate whether discrepancies are application-specific

• Look for consensus results across multiple antibodies

• Use orthogonal methods (e.g., mass spectrometry, mRNA analysis) to resolve antibody-based discrepancies

• Review the validation category of each antibody according to standardized assessment criteria

• Consider biological variables such as post-translational modifications or protein-protein interactions that might affect epitope recognition

How can CSNK2A1 antibodies be used to study protein-protein interactions?

For protein interaction studies with CSNK2A1:

• Co-immunoprecipitation (Co-IP): Use validated CSNK2A1 antibodies to precipitate protein complexes, followed by Western blot detection of potential interaction partners

• Proximity ligation assay (PLA): Visualize in situ protein interactions with spatial resolution

• Pull-down experiments: Compare results between wild-type and knockout samples to distinguish specific versus non-specific interactions

• Mass spectrometry following IP: Identify novel interaction partners in an unbiased manner

• Controls: Include IgG controls and knockout cells to establish specificity thresholds

What considerations are important when studying CSNK2A1 in disease models?

When investigating CSNK2A1 in disease contexts:

• Tissue selection: Human breast cancer and colon cancer tissues have shown positive staining for CSNK2A1 in IHC

• Antigen retrieval: Optimize using either TE buffer pH 9.0 or citrate buffer pH 6.0

• Antibody selection: Choose antibodies validated in tissues similar to your disease model

• Controls: Include both positive controls (tissues with known CSNK2A1 expression) and negative controls

• Quantification: Apply digital pathology approaches for objective assessment of expression levels

• Comparative analysis: Consider CSNK2A1 expression in relation to disease progression or treatment response

• Molecular context: Examine relationship to known disease mechanisms, particularly in autistic spectrum disorders with CSNK2A1 mutations

How can I investigate post-translational modifications of CSNK2A1?

To study post-translational modifications of CSNK2A1:

• Immunoprecipitation: Use validated CSNK2A1 antibodies to isolate the protein

• Western blot analysis: Probe with antibodies specific for modifications (phosphorylation, ubiquitination, etc.)

• Mass spectrometry: Perform proteomic analysis of immunoprecipitated CSNK2A1 to identify modification sites

• Functional studies: Correlate modifications with CSNK2A1 kinase activity, localization, stability, or protein interactions

• Inhibitor studies: Treat cells with modification-specific inhibitors to establish functional relationships

• Mutational analysis: Compare wild-type CSNK2A1 with mutants at specific modification sites

• Controls: Include appropriate treatment controls (phosphatase inhibitors, deubiquitinase inhibitors, etc.)