Phospho-LCK (S540) Antibody

What is LCK and what role does S540 phosphorylation play in T cell signaling?

LCK (Lymphocyte-specific protein tyrosine kinase) is essential for T-lymphocyte activation and differentiation, serving as a critical upstream kinase in T cell receptor (TCR) signaling . While phosphorylation at Tyr505 downregulates LCK's catalytic activity and phosphorylation at Tyr394 increases its activity , the S540 phosphorylation represents an additional regulatory site that contributes to fine-tuning LCK function in TCR signaling cascades . Understanding S540 phosphorylation provides additional insight into the complex regulation of this crucial signaling protein.

How does Phospho-LCK (S540) antibody differ from other LCK phosphorylation site antibodies?

The Phospho-LCK (S540) antibody specifically recognizes LCK when phosphorylated at serine residue 540, unlike antibodies targeting phosphorylation at Tyr505 or Tyr394 . This specificity allows researchers to distinguish between different regulatory states of LCK. For example, Phospho-LCK (Y505) antibodies detect the inhibitory phosphorylation site, whereas the S540 antibody recognizes a distinct regulatory modification, enabling more comprehensive analysis of LCK's phosphorylation status during T cell activation .

What are the validated applications for Phospho-LCK (S540) antibody?

According to available data, Phospho-LCK (S540) antibody has been validated for:

While these applications have been validated, researchers should perform their own optimization for specific experimental conditions. The antibody may potentially be useful for other applications such as immunohistochemistry or flow cytometry, though additional validation would be required .

What are the optimal conditions for using Phospho-LCK (S540) antibody in Western blotting experiments?

For optimal Western blotting results with Phospho-LCK (S540) antibody:

• Use dilutions between 1:500 and 1:2000 as recommended

• Prepare samples in appropriate lysis buffer containing phosphatase inhibitors (e.g., sodium orthovanadate) to preserve phosphorylation status

• Run samples under reducing conditions on SDS-PAGE gels

• Transfer to PVDF or nitrocellulose membranes

• Block with 5% BSA in TBST rather than milk, as milk contains phosphoproteins that may interfere with detection

• Incubate with primary antibody overnight at 4°C for optimal binding

• Use appropriate HRP-conjugated secondary antibodies (anti-rabbit IgG for polyclonal antibodies)

For comparison, protocols for other phospho-specific LCK antibodies involve similar approaches but may require different dilutions (e.g., Phospho-Lck Y505 is typically used at 1:1000) .

How can I optimize T cell stimulation to study S540 phosphorylation dynamics?

To effectively study S540 phosphorylation dynamics:

• For in vitro T cell activation, consider these evidence-based approaches:

  • Anti-CD3ε antibody stimulation (5 μg/mL at 37°C)

  • Pervanadate treatment (1 mM for 10 minutes) to inhibit phosphatases

  • H₂O₂ treatment (2 mM for 2 minutes) to modulate redox-sensitive phosphorylation

  • Superantigen stimulation (e.g., SEE at 30 ng/mL)

• Include appropriate time points (typically 1-30 minutes) to capture phosphorylation kinetics

• Lyse cells in buffer containing phosphatase inhibitors (4 mM NaVO₃, 40 mM NaF) to preserve phosphorylation status

• Consider parallel assessment of multiple phosphorylation sites (Y505, Y394, S540) to understand their interrelationships

What controls should I include when working with Phospho-LCK (S540) antibody?

Robust experimental design requires appropriate controls:

• Positive controls: Jurkat T cells stimulated with pervanadate or H₂O₂ show detectable LCK phosphorylation

• Negative controls:

  • Unstimulated T cells (resting state)

  • Samples treated with phosphatase

  • Isotype control antibody to assess non-specific binding

• Specificity controls:

  • Blocking peptide competition assay using the immunizing peptide

  • siRNA knockdown of LCK to confirm band specificity

  • Comparing reactivity across species (human, mouse, rat) as specified in product information

• Loading controls: β-actin or other housekeeping proteins to normalize protein loading

How can I use Phospho-LCK (S540) antibody to distinguish between different T cell activation states?

Advanced experimental approaches include:

• Temporal analysis: Monitor S540 phosphorylation alongside Y505 and Y394 phosphorylation during T cell activation to create a phosphorylation signature profile specific to different T cell stimuli

• Stimulus comparison:

  • Compare TCR-mediated versus cytokine-mediated phosphorylation patterns

  • Analyze differences between strong TCR stimulation (which induces transient calcium elevation) versus weak stimulation (which generates sustained calcium oscillations)

• Single-cell analysis:

  • Combine phospho-flow cytometry with other cellular markers to identify cell subpopulations with distinct phosphorylation profiles

  • Correlate with functional outcomes (e.g., cytokine production, proliferation)

• High-throughput methodologies:

  • Combine with peptide library screens similar to those described for other phosphorylation sites to identify substrate preferences

  • Integrate with proteomics approaches to map complex signaling networks

How can molecular dynamics simulations enhance our understanding of LCK S540 phosphorylation?

Molecular dynamics approaches can reveal crucial mechanistic insights:

• Similar to methods described for other LCK phosphorylation sites, researchers can conduct 500+ ns simulations of the LCK kinase domain with modeled S540 phosphorylation

• Key simulation parameters based on published protocols:

  • Use crystal structures of LCK kinase domain in active conformation as starting coordinates

  • Model substrate peptides based on known LCK targets

  • Include ATP, Mg²⁺ ions, and water molecules in the active site

  • Cap polypeptide chains with N-acetyl and N-methylcarboxamide groups

• Analyze simulations to determine:

  • Conformational changes induced by S540 phosphorylation

  • Potential electrostatic interactions affected by this modification

  • Influence on substrate binding and catalytic activity

• Compare with simulations of other phosphorylation states (Y394, Y505) to develop a comprehensive model of LCK regulation

What are common challenges when working with Phospho-LCK (S540) antibody and how can they be addressed?

How does sample preparation affect Phospho-LCK (S540) detection?

Sample preparation is critical for preserving phosphorylation status:

• Lysis buffer composition: Use buffers containing effective phosphatase inhibitor combinations. Published protocols recommend:

  • 2-4% NP40 or equivalent detergent

  • 50 mM Tris-HCl

  • 150 mM NaCl

  • 10 mM EDTA

  • 4 mM NaVO₃

  • 40 mM NaF (pH 7.4)

• Temperature control: Maintain samples at 4°C during processing to minimize phosphatase activity

• Timing considerations:

  • Process samples immediately after stimulation

  • For time-course experiments, use rapid sample quenching methods

  • Consider flash-freezing samples if immediate processing isn't possible

• Storage conditions: Store antibody according to manufacturer recommendations (typically at -20°C or -80°C, avoid repeated freeze-thaw cycles)

How can I optimize detection of multiple LCK phosphorylation sites in a single experiment?

For comprehensive LCK phosphorylation analysis:

• Sequential immunoblotting:

  • Strip and reprobe membranes with different phospho-specific antibodies

  • Carefully control stripping conditions to preserve epitopes

  • Consider running duplicate gels if multiple phospho-sites must be analyzed simultaneously

• Multiplex flow cytometry:

  • Use differently conjugated secondary antibodies for each phospho-specific primary

  • Employ careful titration to prevent antibody interference

  • Validate specificity with appropriate controls

• Imaging techniques:

  • For cellular localization studies, use appropriate fixation methods that preserve phospho-epitopes

  • Consider TIRF-SIM imaging for TCR clustering studies as described in the literature

  • Optimize immunofluorescence protocols for each phospho-antibody separately before attempting multiplex detection