CSK Antibody

What is CSK and what is its functional role in cellular signaling?

CSK (C-terminal Src kinase, also known as protein-tyrosine kinase CYL) is a non-receptor tyrosine kinase with a molecular mass of 50 kDa that belongs to the protein kinase superfamily. Structurally, it contains SH3 and SH2 domains in its N-terminus and a kinase domain in its C-terminus. CSK functions primarily as a negative regulator of Src-family kinases (SFKs), playing crucial roles in multiple physiological functions via signaling pathways that control cell proliferation, differentiation, adhesion, and migration . In T cells specifically, CSK functions as a critical regulator that helps establish the T cell receptor (TCR) signaling threshold and controls affinity recognition .

What applications are CSK antibodies commonly used for in research?

CSK antibodies are utilized across multiple research applications, with the most common being Western Blot (WB), Immunofluorescence (IF)/Immunocytochemistry (ICC), and Enzyme-Linked Immunosorbent Assay (ELISA) . In Western blotting, CSK antibodies enable detection of the 50 kDa CSK protein, while immunofluorescence applications allow researchers to visualize the cytosolic localization of CSK. These antibodies have demonstrated reactivity with human and mouse samples, making them suitable for comparative studies across these species .

What are the optimal storage conditions for CSK antibodies?

CSK antibodies should typically be stored at -20°C for long-term storage, where they remain stable for approximately one year after shipment . For antibodies in PBS buffer containing sodium azide, storage at 4°C is recommended, with explicit instructions not to freeze . It's important to note that while aliquoting is unnecessary for -20°C storage of some formulations, it may be beneficial for antibodies stored at 4°C to avoid repeated freezing and thawing cycles that could compromise antibody integrity .

What are the recommended dilutions for CSK antibodies in different applications?

The recommended dilution ranges for CSK antibodies vary by application:

It is important to note that these are general recommendations, and researchers should titrate the antibody in each specific testing system to obtain optimal results. Sensitivity may also be sample-dependent, requiring optimization for different cellular contexts .

What cell types are suitable as positive controls for CSK antibody validation?

Based on available validation data, several cell lines have been confirmed to express detectable levels of CSK and can serve as positive controls:

These cell lines represent diverse tissue origins and species (human and mouse), providing researchers with multiple options for experimental validation .

How can researchers optimize CSK antibody performance in immunocytochemistry applications?

For optimal immunocytochemistry results with CSK antibodies, researchers should consider using a co-staining approach to aid in interpretation. For example, validated protocols have successfully combined CSK antibody staining (visualized in green) with phalloidin to decorate the actin cytoskeleton (visualized in red) and DAPI for nuclear staining (blue) . This approach provides contextual information about CSK localization relative to cytoskeletal structures and the nucleus. The recommended dilution range of 1:500-1:2000 serves as a starting point, though optimization may be necessary depending on the specific cell type and fixation method employed .

How does inhibition of CSK affect T cell receptor signaling dynamics?

Studies utilizing a mutated form of CSK (CskAS) that can be specifically inhibited by the small molecule 3-iodo-benzyl-PP1 (3-IB-PP1) have provided insights into CSK's role in T cell receptor signaling. Inhibition of CSK during TCR stimulation results in stronger and more prolonged TCR signaling, characterized by extended phosphorylation of key signaling molecules including ZAP-70, LAT, and PLC-γ1 .

The effect on signal termination is particularly noteworthy: while CSK inhibition delays signal downregulation, it does not completely prevent signal attenuation over time. This indicates that CSK plays only a partial role in signal termination, with other negative regulatory mechanisms contributing to the eventual termination of TCR signaling, albeit more slowly in the absence of CSK activity . Researchers investigating CSK's role in signaling duration should therefore consider complementary mechanisms when interpreting results.

What phosphorylation changes occur when CSK activity is inhibited in T cells?

When CSK activity is inhibited, complex changes in phosphorylation patterns can be observed. Intriguingly, inhibition of CSK leads to a 3-4 fold upregulation of activating phosphorylation sites on Src family kinases, while the inhibitory tail Y505 phosphorylation (the direct target of CSK) does not decrease as substantially as might be expected .

This observation suggests a model where Lck (a Src family kinase crucial for T cell activation) is actively phosphorylated by CSK, but a substantial proportion of the phosphorylated Y505 residue is either inaccessible for dephosphorylation by CD45 or is dephosphorylated over a longer timescale. Researchers should consider this complexity when designing experiments to study CSK's regulatory role, as the relationship between CSK inhibition and downstream phosphorylation events is not straightforward .

How can researchers distinguish CSK's effects on basal signaling versus induced signaling thresholds?

To differentiate between CSK's roles in basal signaling and in setting activation thresholds, researchers can implement experimental designs that compare responses to stimuli of varying strengths with and without CSK inhibition. Studies have demonstrated that inhibition of CSK particularly enhances T cell responses to weak cognate agonists .

A dose-titration approach with CSK inhibitors reveals that even very small increases in Src family kinase activity (resulting from minimal CSK inhibition) can significantly potentiate T cell responses to weak agonists. This indicates that CSK functions not only to maintain low basal signaling but also plays a crucial role in establishing the threshold at which T cells respond to stimuli. Researchers investigating CSK's functional roles should design experiments that can distinguish between these distinct regulatory functions .

What disease contexts are relevant for CSK-focused research?

CSK may have significant relevance to multiple disease states, as indicated by its association with several pathological conditions. Research examining CSK's role in disease pathogenesis has implicated it in:

• Adenocarcinoma

• Alzheimer's disease

• Asthma

• Cardiovascular diseases

• Diabetes mellitus and its complications

• Edema

• Esophageal neoplasms

• Conditions with genetic predisposition components

These associations suggest that CSK may play roles in diverse physiological systems beyond immune regulation, including potential functions in metabolic, cardiovascular, and neurodegenerative contexts. Researchers studying these disease areas should consider incorporating CSK-focused investigations into their experimental designs.

How might CSK-targeting approaches be developed for therapeutic applications?

The finding that small molecule inhibition of CSK can enhance T cell responses, particularly to weak agonists, suggests potential therapeutic applications. CSK inhibition represents a possible approach for fine-tuning immune responses in contexts where enhanced T cell reactivity would be beneficial, such as cancer immunotherapy or vaccine development .

Research supporting this therapeutic direction demonstrates that CSK inhibition enhances T cell proliferation and prolongs signaling, particularly in response to weak cognate antigens that might not normally activate T cells sufficiently. Future therapeutic development would require identifying compounds that can selectively inhibit wild-type CSK in physiological contexts, followed by in vivo testing in appropriate animal models . Researchers exploring translational applications should focus on developing inhibitors with appropriate selectivity and pharmacokinetic properties.

What are important considerations when interpreting phosphorylation status in CSK studies?

When studying CSK's effects on phosphorylation events, researchers should be mindful of several complexities. The relationship between CSK inhibition and changes in phosphorylation of its target sites (such as Y505 on Lck) is not straightforward. Studies have shown that even when CSK is inhibited, the decrease in inhibitory phosphorylation may be smaller than expected, yet still result in significant functional changes .

Additionally, researchers should consider the digital (all-or-none) nature of some downstream responses, such as ERK phosphorylation, compared to the more analog and heterogeneous nature of signal downregulation. When designing experiments to study the impact of CSK on phosphorylation dynamics, time-course analyses are essential, as differences in signal duration may be as important as differences in signal magnitude .

What controls should be included when using CSK antibodies for protein detection?

When utilizing CSK antibodies for protein detection, researchers should implement multiple controls to ensure specificity and reliability:

• Positive controls: Include cell lines known to express CSK, such as Jurkat, HeLa, NIH/3T3, or RAW 264.7 cells for Western blotting applications .

• Loading controls: Use appropriate housekeeping proteins to normalize for total protein loading.

• Molecular weight verification: Confirm that detected bands appear at the expected molecular weight of approximately 50 kDa .

• Antibody specificity control: Consider using CSK-knockout or knockdown samples where available to confirm the specificity of the observed signal.

• Application-specific controls: For immunofluorescence applications, include controls for secondary antibody specificity and consider counterstaining with phalloidin and DAPI to provide cellular context for the observed staining pattern .