CLK1 Antibody

What is CLK1 and what cellular functions does it regulate?

CLK1 (CDC-like kinase 1) is a dual-specificity protein kinase that plays crucial roles in RNA splicing regulation through phosphorylation of SR proteins. It has a calculated molecular weight of 57 kDa (484 amino acids), although it typically appears at approximately 62 kDa in Western blots due to post-translational modifications . Recent research has revealed its involvement in kinetochore function and cell cycle control, particularly in mitosis regulation . Additionally, CLK1 has emerged as a potential therapeutic target for trypanosomatid parasitic diseases and appears to play a role in HIV-1 gene expression and latency .

Methodological approach: To study CLK1 functions, researchers often employ both gain-of-function (overexpression) and loss-of-function (RNAi knockdown, CRISPR-Cas9 knockout, or chemical inhibition) strategies. Phenotypic analyses focusing on cell cycle progression, RNA splicing patterns, and protein-protein interactions can reveal specific functional domains.

What applications are CLK1 antibodies typically used for?

CLK1 antibodies are utilized across multiple experimental applications with varying dilution requirements:

Methodological approach: For optimal results, researchers should perform antibody titration experiments for each application and biological system. For example, in Western blotting, a dilution series can determine the optimal antibody concentration that maximizes specific signal while minimizing background.

What species reactivity can be expected from available CLK1 antibodies?

Commercial CLK1 antibodies show varying species reactivity profiles:

Methodological approach: When investigating CLK1 in non-validated species, researchers should first confirm cross-reactivity through a pilot Western blot experiment with positive controls from validated species alongside the test sample. Sequence alignment analysis between human CLK1 and the target species can predict potential cross-reactivity.

How should sample preparation be optimized for CLK1 detection?

CLK1 detection requires careful sample preparation to preserve protein integrity and phosphorylation state:

• Cell lysis buffer selection: Use buffers containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate) to preserve phosphorylation states.

• Positive control selection: COLO 320, HeLa, and HepG2 cells have been validated for CLK1 Western blot detection .

• Denaturation conditions: Standard reducing conditions (β-mercaptoethanol or DTT) with heating at 95°C for 5 minutes are typically sufficient.

Methodological approach: For phosphorylation-specific studies, researchers should compare multiple lysis protocols and immediately process samples on ice to minimize dephosphorylation by endogenous phosphatases.

What storage conditions maintain CLK1 antibody stability?

For optimal CLK1 antibody performance and longevity:

• Storage temperature: Store at -20°C. Most CLK1 antibodies remain stable for at least one year after shipment when properly stored .

• Buffer composition: Typical storage buffers contain PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Aliquoting: While aliquoting is not strictly necessary for -20°C storage of glycerol-containing preparations, it can prevent freeze-thaw cycles that may compromise antibody activity .

Methodological approach: Researchers should maintain a stock-working solution system, where the original antibody remains at -20°C while a working aliquot is kept at 4°C for up to two weeks.

What controls are essential for validating CLK1 antibody specificity?

Comprehensive validation requires multiple controls:

• Positive controls: Use cell lines with confirmed CLK1 expression, such as COLO 320, HeLa, or HepG2 cells .

• Negative controls: Include:

  • Primary antibody omission

  • Isotype control (rabbit IgG)

  • CLK1 knockdown/knockout samples

  • Peptide competition assay using the immunogen peptide

• Molecular weight verification: Confirm detection at the expected 62 kDa band .

Methodological approach: For definitive validation, researchers should generate CLK1 knockdown or knockout cell lines using shRNA or CRISPR-Cas9 technology. This allows for direct comparison of antibody signal between wild-type and CLK1-deficient samples.

How can immunoprecipitation protocols be optimized for CLK1 studies?

For successful CLK1 immunoprecipitation:

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

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Binding conditions: Incubate overnight at 4°C with gentle rotation.

• Validation: COLO 320 cells have been validated for successful CLK1 immunoprecipitation .

Methodological approach: When investigating protein-protein interactions, researchers should consider crosslinking approaches to capture transient interactions. Comparing native versus denaturing elution conditions can provide insights into direct versus indirect interactions.

What methodologies can detect CLK1 kinase activity in experimental systems?

Several approaches can assess CLK1 enzymatic function:

• In vitro kinase assays: Using recombinant CLK1 with known substrates (SR proteins) and measuring phosphate incorporation.

• Cellular phosphorylation: Monitoring phosphorylation state of known CLK1 substrates after CLK1 manipulation.

• Inhibitor studies: Measuring IC50 values of compounds against recombinant TbCLK1 compared to human CLK1, which has shown correlation with cellular activity (Pearson's correlation r = 0.68) .

Methodological approach: Researchers investigating CLK1 activity should employ a multi-faceted approach combining in vitro biochemical assays with cellular readouts. Using phospho-specific antibodies against known CLK1 substrates can provide functional validation in cellular contexts.

How can researchers establish reliable CLK1 overexpression or knockdown models?

For manipulating CLK1 expression levels:

• Overexpression systems:

  • Transient transfection with CLK1 expression vectors

  • Stable cell lines with inducible CLK1 expression

  • Viral delivery systems for hard-to-transfect cells

• Knockdown/knockout approaches:

  • siRNA/shRNA for transient or stable knockdown

  • CRISPR-Cas9 for genetic knockout

  • Chemical inhibition with CLK1-specific inhibitors

Methodological approach: Research indicates that CLK1 overexpression can impair parasite fitness without affecting cell cycle progression, while CLK1 knockdown or inhibition results in cell cycle arrest at G2/M phase with enlarged nuclei . For comprehensive phenotypic analysis, researchers should employ both approaches and validate expression changes by Western blot.

What considerations are important when designing immunofluorescence experiments with CLK1 antibodies?

For optimal CLK1 immunofluorescence:

• Fixation method: Compare paraformaldehyde (preserves structure) versus methanol (better epitope exposure) fixation.

• Permeabilization: Use 0.1-0.5% Triton X-100 or 0.1% saponin to access intracellular epitopes.

• Blocking: BSA (1-5%) in PBS with 0.1% Tween-20 reduces background.

• Controls: Include secondary-only controls and CLK1-depleted samples.

Methodological approach: For colocalization studies, researchers should perform sequential immunostaining with validated markers of nuclear speckles (SC35) or kinetochores (CENP proteins) to investigate CLK1's functional compartmentalization during different cell cycle phases.

How is CLK1 involved in kinetochore function and cell cycle regulation?

Recent research has established CLK1 as a critical component of the kinetochore complex:

• In Trypanosoma brucei, CLK1 (also known as KKT10) is essential for mitosis .

• Chemical inhibition or genetic depletion of CLK1 leads to:

  • G2/M cell cycle arrest

  • Chromosome mis-segregation

  • Enlarged nuclei

  • Compromised kinetochore recruitment

• CLK1 overexpression in T. brucei confers resistance to the G2/M cell cycle arrest induced by amidobenzimidazole inhibitors .

Methodological approach: Researchers investigating CLK1's role in cell cycle regulation should combine live-cell imaging with fixed-cell immunofluorescence at defined cell cycle stages. Time-course experiments following CLK1 inhibition or depletion can reveal the temporal sequence of kinetochore assembly defects and downstream consequences.

What is the role of CLK1 in HIV-1 gene expression and latency reversal?

Emerging evidence points to CLK1 as a regulator of HIV-1 gene expression:

• Reduced CLK1 expression enhances the response to latency reversing agents (LRAs) .

• CLK1 depletion increases the frequency of HIV-1 expressing cells upon doxycycline induction in the CEM-HIV* cell line model .

• The combination of CLK1 depletion with the PKC activator prostratin further increases the frequency of GagGFP+ cells .

Methodological approach: For HIV latency studies, researchers should use established latency cell models (e.g., CEM-HIV* cells) and measure viral reactivation using reporter genes or direct viral RNA/protein quantification. Flow cytometry can determine whether CLK1 manipulation affects the percentage of responding cells or the expression level within individual cells.

How can CLK1 be targeted for therapeutic development in parasitic diseases?

CLK1 represents a promising drug target for trypanosomatid parasites:

• T. brucei CLK1 differs from human CLK1 at position C215 in the ATP binding pocket, providing selectivity for inhibitor development .

• Amidobenzimidazole compounds with a Michael acceptor form an irreversible bond with C215, showing up to 90-fold selectivity for TbCLK1 over human CLK1 .

• Strong correlation exists between TbCLK1 enzyme inhibition and parasite killing (Pearson's correlation r = 0.68) .

• Co-crystal structure of CLK1 in complex with AB1 (inhibitor) is available (PDB: 6Q2A) .

Methodological approach: Drug discovery efforts should leverage the CLK1 crystal structure for structure-based design of selective inhibitors. Both covalent (targeting C215) and non-covalent approaches can be pursued, with biochemical assays measuring kinase inhibition followed by cellular assays assessing parasite growth inhibition.

What methods can determine CLK1's impact on alternative splicing patterns?

As a regulator of SR proteins, CLK1 influences RNA splicing:

• Transcriptome-wide approaches:

  • RNA-seq following CLK1 manipulation identifies global splicing changes

  • Alternative splicing event analysis using computational tools (MISO, rMATS)

  • Direct comparison of exon inclusion/exclusion ratios

• Targeted approaches:

  • RT-PCR of specific alternatively spliced transcripts

  • Minigene splicing reporters

Methodological approach: Researchers should combine RNA-seq with validation of individual splicing events using RT-PCR. CLK1 inhibition or depletion experiments can be performed with time-course analyses to distinguish direct from indirect effects on the splicing machinery.

How can CLK1's role in cancer progression be investigated?

CLK1 has emerging implications in cancer biology:

• Recent machine learning approaches have identified CLK1 as a potential immune cell marker in osteosarcoma with prognostic significance in disease progression .

• Research methodologies to investigate this connection include:

  • Cancer tissue microarray analysis with CLK1 antibodies

  • Correlation of CLK1 expression with clinical outcomes

  • Functional studies in cancer cell lines and animal models

Methodological approach: Researchers should analyze CLK1 expression across cancer types using tissue microarrays and correlate with clinical parameters. Functional studies in cell lines can assess the impact of CLK1 modulation on hallmark cancer phenotypes (proliferation, migration, invasion, drug resistance).

Why might there be a discrepancy between calculated and observed molecular weights of CLK1?

CLK1 has a calculated molecular weight of 57 kDa (484 amino acids) but typically appears at approximately 62 kDa in Western blots . This discrepancy may arise from:

• Post-translational modifications: CLK1 undergoes autophosphorylation and may have other modifications affecting mobility.

• Protein structure: Certain domains or amino acid compositions can affect SDS binding and alter migration.

• Gel composition: Percentage of acrylamide and buffer systems can influence apparent molecular weight.

Methodological approach: To confirm band identity, researchers should compare wild-type samples with CLK1 knockdown/knockout controls. Additionally, treatment with phosphatase can determine if the molecular weight shift is due to phosphorylation.

What strategies can minimize non-specific binding when using CLK1 antibodies?

To improve signal-to-noise ratio:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Increase blocking time and concentration for high background

  • Add 0.1-0.5% Tween-20 to reduce hydrophobic interactions

• Antibody incubation optimization:

  • Titrate antibody concentration

  • Extend incubation time with more dilute antibody

  • Try different diluents (TBS vs. PBS, with/without detergents)

• Washing stringency:

  • Increase wash duration and volume

  • Add higher salt concentration to wash buffers

Methodological approach: Perform a systematic optimization by testing a matrix of conditions, varying one parameter at a time. Include appropriate controls (primary antibody omission, isotype controls) to identify the source of background signal.

How can researchers address variability in CLK1 detection between experiments?

For consistent CLK1 detection:

• Standardize lysate preparation:

  • Use consistent cell density and harvest conditions

  • Standardize lysis buffer and protein determination method

  • Include protease and phosphatase inhibitors

• Internal controls:

  • Always include loading controls (β-actin, GAPDH)

  • Run a common reference sample across all blots for normalization

  • Consider multiplexing with differently colored secondary antibodies

• Technical considerations:

  • Maintain consistent transfer conditions

  • Use automated image capture with fixed exposure settings

  • Apply quantitative analysis with appropriate software

Methodological approach: Implement a laboratory standard operating procedure (SOP) for all CLK1 experiments. For critical comparisons, process all samples simultaneously rather than in separate experiments.

What approaches can resolve contradictory results in CLK1 studies?

When facing conflicting data:

• Antibody validation:

  • Confirm specificity with knockdown/knockout controls

  • Try alternative antibodies targeting different epitopes

  • Validate with orthogonal methods (mass spectrometry)

• Cell type considerations:

  • CLK1 function may vary by cell type (compare results across validated cell lines like COLO 320, HeLa, and HepG2)

  • Consider cell-cycle dependent effects

• Experimental conditions:

  • Control for environmental variables (confluence, passage number)

  • Test kinetic responses rather than single timepoints

  • Consider compensation mechanisms (redundancy with other CLK family members)

Methodological approach: When results conflict with published findings, systematically test key variables that might explain differences. Direct communication with authors of conflicting studies can often clarify methodological differences not detailed in publications.

How can CLK1 protein degradation during sample preparation be prevented?

To maintain CLK1 integrity:

• Buffer optimization:

  • Include fresh protease inhibitor cocktail

  • Maintain cold temperature throughout processing

  • Consider adding specific inhibitors for proteases abundant in your sample type

• Processing speed:

  • Minimize time between cell lysis and protein denaturation

  • Process samples in small batches to reduce handling time

  • Consider direct lysis in SDS sample buffer for immediate denaturation

• Storage considerations:

  • Avoid freeze-thaw cycles of protein samples

  • Add 50% glycerol for long-term storage at -20°C

  • Consider aliquoting samples for single use

Methodological approach: For problematic samples, compare multiple extraction protocols side-by-side. Test the stability of CLK1 in your specific sample type by analyzing aliquots after different storage durations and conditions.

How can structural biology approaches advance CLK1 inhibitor development?

Structural studies provide critical insights for targeted CLK1 inhibitor design:

• X-ray crystallography has revealed:

  • T. brucei CLK1 in complex with covalent aminobenzimidazole inhibitor AB1 (PDB: 6Q2A)

  • Key differences in the ATP binding pocket between parasite and human CLK1

• Structure-based drug design opportunities:

  • Exploiting the C215 residue unique to T. brucei CLK1 for selectivity

  • Designing non-covalent inhibitors through fragment-based screens

  • Structure-guided optimization to improve selectivity and pharmacokinetics

Methodological approach: Researchers should combine in silico docking studies with biochemical validation using recombinant CLK1 proteins. Critical structure-activity relationship analyses can guide medicinal chemistry optimization of lead compounds.

What high-throughput methods can accelerate CLK1 research?

Advanced technologies enabling CLK1 research at scale:

• CRISPR screening:

  • Genome-wide screens for synthetic lethality with CLK1 inhibition

  • Targeted screens of splicing regulators to identify functional relationships

• Chemical biology approaches:

  • Small molecule microarrays for inhibitor discovery

  • Activity-based protein profiling for target engagement confirmation

• Automated microscopy:

  • High-content imaging to assess cellular phenotypes

  • Live-cell tracking of CLK1 dynamics during cell cycle

Methodological approach: Researchers should leverage multi-parametric assays that can simultaneously measure multiple endpoints (e.g., cell cycle, apoptosis, splicing changes) following CLK1 manipulation, enabling more comprehensive phenotypic characterization.

How might single-cell technologies reveal heterogeneity in CLK1 function?

Single-cell approaches offer new insights into CLK1 biology:

• Single-cell RNA-seq can reveal:

  • Cell-to-cell variability in CLK1 expression

  • Correlation between CLK1 levels and alternative splicing patterns

  • Subpopulation-specific responses to CLK1 inhibition

• Single-cell protein analysis:

  • Mass cytometry (CyTOF) to correlate CLK1 with other signaling pathways

  • Microfluidic approaches for kinase activity measurements

Methodological approach: For HIV-1 latency research, single-cell approaches are particularly valuable as they can distinguish between increased expression per cell versus increased frequency of expressing cells following CLK1 depletion . Flow cytometry analysis of reporter gene expression in individual cells can quantify both the percentage of positive cells and expression intensity.

What is the potential of CLK1 as a biomarker in disease diagnostics?

Emerging evidence suggests CLK1 diagnostic applications:

• Cancer biomarker potential:

  • Machine learning has identified CLK1 as a prognostic marker in osteosarcoma

  • Immunohistochemical detection in tumor tissues could stratify patients

• Infectious disease applications:

  • CLK1 activity measurement in parasite isolates could predict drug resistance

  • CLK1 inhibition sensitivity might guide personalized therapeutic approaches

Methodological approach: Researchers should conduct retrospective analyses of patient samples with known outcomes to evaluate CLK1's predictive value. Prospective studies can then validate findings and establish clinically relevant cutoffs for diagnostic use.

How can multi-omics integration advance understanding of CLK1 biology?

Integrative approaches to comprehensively map CLK1 function:

• Combined transcriptomics and proteomics:

  • Correlate splicing changes with protein isoform expression

  • Identify feedback mechanisms in CLK1 regulatory networks

• Phosphoproteomics:

  • Map CLK1 substrates through differential phosphorylation analysis

  • Identify regulatory phosphorylation sites on CLK1 itself

• Interactomics:

  • BioID or APEX proximity labeling to map CLK1 protein interactions

  • Temporal interaction changes during cell cycle progression