LCK (Ab-394) Antibody

What is the LCK (Ab-394) Antibody and what epitope does it recognize?

The LCK (Ab-394) Antibody is a rabbit polyclonal antibody generated against a specific peptide sequence around amino acids 392-396 (N-E-Y-T-A) derived from Human Lck. This region contains the critical tyrosine residue (Y394) that, when phosphorylated, indicates the activated state of Lck . The antibody was produced by immunizing rabbits with a synthetic peptide conjugated to KLH and subsequently purified using affinity chromatography with the epitope-specific peptide . This purification process ensures high specificity for the target epitope region of Lck.

What applications has the LCK (Ab-394) Antibody been validated for?

The LCK (Ab-394) Antibody has been primarily validated for Western blotting (WB) applications . This application is particularly useful for detecting Lck protein expression levels and, in combination with other antibodies, can help determine the relative proportions of differently phosphorylated Lck forms in experimental samples.

When using this antibody for Western blotting, researchers should consider the following methodological aspects:

• Sample preparation: Cell lysates should be prepared using buffers that preserve protein phosphorylation states if studying Lck activation

• Protein denaturation: Complete denaturation is essential for accurate epitope exposure

• Blocking conditions: Typically 5% BSA in TBST works well for phosphoprotein detection

• Antibody dilution: Optimize based on signal-to-noise ratio in your specific experimental system

• Detection method: Both chemiluminescence and fluorescence-based detection systems are compatible

While the product information specifically validates Western blotting, researchers investigating T cell signaling pathways often explore additional applications such as immunoprecipitation, immunofluorescence, or flow cytometry after performing their own validation studies.

What is the species reactivity profile of the LCK (Ab-394) Antibody?

The LCK (Ab-394) Antibody demonstrates cross-reactivity with Lck from multiple species, specifically Human, Mouse, and Rat samples . This broad species reactivity makes the antibody valuable for comparative studies across different experimental models. The cross-reactivity stems from the high conservation of the epitope region (amino acids 392-396) across these species.

When using this antibody with samples from species other than human, mouse, or rat, researchers should perform preliminary validation experiments to confirm reactivity. Additionally, when working with complex tissue samples or mixed cell populations, additional controls may be necessary to confirm specificity within the particular experimental context.

How should the LCK (Ab-394) Antibody be stored and handled for optimal results?

For optimal stability and performance, the LCK (Ab-394) Antibody should be stored at -20°C for long-term preservation . For short-term use (typically within 1-2 weeks), storage at 4°C is acceptable. The antibody is supplied at a concentration of 1.0 mg/mL in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, containing 150mM NaCl, 0.02% sodium azide, and 50% glycerol .

Best practices for handling include:

• Aliquoting upon first thaw to minimize freeze-thaw cycles

• Maintaining cold chain during experimental procedures

• Using clean pipette tips to prevent contamination

• Centrifuging briefly before opening to collect liquid at the bottom of the tube

• Avoiding prolonged exposure to light, particularly if the antibody is conjugated

• Checking for signs of precipitation before use, which may indicate denaturation

Proper storage and handling significantly impact experimental reproducibility and antibody longevity, potentially extending the useful life of the reagent from months to years.

How can the LCK (Ab-394) Antibody be used to investigate different phosphorylation states of Lck?

While the LCK (Ab-394) Antibody itself recognizes total Lck protein rather than specific phosphorylation states, it can be strategically used in combination with phospho-specific antibodies to investigate the different Lck activation states. Research has identified four major forms of Lck based on phosphorylation at Y394 (activation site) and Y505 (inhibitory site) .

To effectively study Lck phosphorylation states, researchers can employ several approaches:

• Immunodepletion experiments: Using anti-pY416 antibodies (which recognize pY394-Lck) to deplete activated Lck, followed by quantification of remaining Lck with the LCK (Ab-394) Antibody

• Sequential immunoprecipitation: Isolating specific Lck forms using phospho-specific antibodies followed by detection with LCK (Ab-394) Antibody

• Phosphatase treatment: Comparing Lck detection before and after phosphatase treatment to estimate the proportion of phosphorylated protein

The table below summarizes the relative proportions of the four Lck forms typically found in unstimulated cells, as determined using these methodologies:

This data indicates that a significant proportion of Lck exists in activated forms even in unstimulated T cells, a finding with important implications for T cell biology research .

What are the optimal experimental conditions for detecting Lck using the LCK (Ab-394) Antibody in Western blotting?

To achieve optimal results when detecting Lck with the LCK (Ab-394) Antibody in Western blotting, researchers should consider several critical experimental parameters:

• Sample preparation:

  • Lyse cells in a buffer containing phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride) to preserve phosphorylation states

  • Include protease inhibitors to prevent protein degradation

  • Maintain cold conditions throughout processing

  • Denature samples completely in reducing SDS buffer (95°C for 5 minutes)

• Gel electrophoresis:

  • Use 8-10% polyacrylamide gels for optimal separation of the 56-59 kDa Lck protein

  • Include molecular weight markers spanning 40-70 kDa range

• Transfer conditions:

  • Wet transfer at 100V for 1 hour or 30V overnight at 4°C is typically effective

  • Use PVDF membranes for better protein retention and lower background

• Blocking and antibody incubation:

  • Block with 5% BSA in TBST (not milk, which contains phosphatases)

  • Incubate with primary antibody at optimized dilution (typically 1:1000) overnight at 4°C

  • Use appropriate HRP-conjugated anti-rabbit secondary antibody (typically 1:5000-1:10000)

• Detection:

  • Use enhanced chemiluminescence (ECL) reagents appropriate for the expected signal intensity

  • For quantitative analysis, consider fluorescence-based secondary antibodies and detection systems

For multi-color Western blotting when examining different phosphorylation states simultaneously, antibodies from different host species should be selected to avoid cross-reactivity of secondary antibodies.

How can researchers quantify the proportion of activated Lck in T cells using the LCK (Ab-394) Antibody?

Quantifying the proportion of activated Lck in T cells requires a strategic approach combining the LCK (Ab-394) Antibody with phospho-specific antibodies. Based on published methodologies, researchers can employ several approaches:

• Immunodepletion method:

  • Use anti-pY416 antibody to immunodeplete pY394-Lck from cell lysates

  • Quantify the remaining Lck using the LCK (Ab-394) Antibody

  • Calculate the proportion of active Lck from the difference between total and remaining Lck

  • This method revealed that >50% of Lck was phosphorylated on Y394 in Jurkat cells and approximately 37% in human naive CD4+ T cells

• Mass spectrometry-based approach:

  • Use isotopically labeled peptide standards corresponding to the Lck tryptic fragment containing Y394

  • Quantify Lck in samples before and after alkaline phosphatase (AP) treatment

  • The difference provides an estimate of pY394-Lck

  • This approach showed approximately 47% of total Lck was in the active form in tested samples

• Comparative immunoblotting:

  • Compare pY394-Lck/total Lck ratio between the experimental sample and a reference sample with known proportion of active Lck

  • This method confirmed approximately 36% activated Lck in normal T cells

These complementary approaches provide robust quantification of activated Lck and reveal that a significant proportion of Lck exists in an activated state even in unstimulated T cells, challenging previous assumptions about Lck activation dynamics.

What controls should be included when using LCK (Ab-394) Antibody in T cell signaling studies?

When designing experiments using the LCK (Ab-394) Antibody for T cell signaling studies, incorporating appropriate controls is essential for result validation and interpretation:

• Positive controls:

  • Jurkat T cell lysates, which express high levels of Lck and contain approximately 56% activated Lck

  • Primary human CD4+ T cells (approximately 37% activated Lck)

  • Recombinant Lck protein (if available) for antibody validation

• Negative controls:

  • Lck-deficient cell lines (e.g., Lck knockout or knockdown cells)

  • Non-T cell lines that do not express Lck (e.g., HEK293 cells)

  • Blocking peptide competition (using the immunogen peptide) to confirm specificity

• Treatment controls:

  • Alkaline phosphatase-treated samples to dephosphorylate Lck

  • T cell receptor stimulation (e.g., anti-CD3/CD28) to induce changes in Lck phosphorylation

  • Src family kinase inhibitors (e.g., dasatinib) to modulate Lck activity

• Antibody controls:

  • IgG isotype control from the same species (rabbit)

  • Sequential probing with phospho-specific antibodies (anti-pY416, anti-pY505)

  • Validation with a second anti-Lck antibody targeting a different epitope

• Loading controls:

  • Housekeeping proteins (e.g., GAPDH, β-actin)

  • Total protein staining (e.g., Ponceau S, REVERT total protein stain)

These controls help ensure specificity, validate activity state measurements, and provide context for interpreting experimental results in T cell signaling studies.

How does Lck activation status detected by LCK (Ab-394) Antibody correlate with T cell functional states?

The activation status of Lck as detected using LCK (Ab-394) Antibody (in combination with phospho-specific antibodies) provides valuable insights into T cell functional states. Research has revealed several important correlations:

• Resting vs. activated T cells:

  • Surprisingly, a significant proportion of Lck exists in an activated form (pY394-Lck) even in unstimulated T cells (approximately 37% in primary human CD4+ T cells)

  • This challenges previous models suggesting that Lck activation occurs primarily after T cell receptor engagement

  • The pre-activated pool of Lck likely facilitates rapid signal initiation upon receptor stimulation

• Different T cell subsets:

  • Naive, memory, and effector T cells may exhibit different proportions of activated Lck

  • The ratio of differently phosphorylated Lck forms (closed-inactive, primed, pY394-active, and DPho-active) correlates with T cell responsiveness

• Pathological conditions:

  • Altered Lck activation patterns are observed in T cell malignancies

  • T-ALL demonstrates LCK-dependency as a therapeutic vulnerability

  • Constitutively active Lck contributes to dysregulated antigen receptor signaling

• T cell development stages:

  • Thymocytes also exhibit the double-phosphorylated form of Lck (DPho-Lck)

  • Changes in Lck phosphorylation status correlate with developmental transitions in the thymus

Researchers can use the LCK (Ab-394) Antibody in combination with other markers to characterize T cell functional states and understand how Lck activation contributes to normal and pathological T cell responses. This information has significant implications for developing therapeutic strategies targeting T cell signaling pathways, as demonstrated by recent work on proteolytic targeting of Lck in T-ALL .

What are the common challenges when using LCK (Ab-394) Antibody and how can they be resolved?

When working with the LCK (Ab-394) Antibody, researchers may encounter several technical challenges that can impact experimental results. Here are common issues and recommended solutions:

• Weak or absent signal:

  • Increase antibody concentration or incubation time

  • Enhance detection sensitivity using amplification systems

  • Verify Lck expression in your sample (Jurkat cells serve as a positive control)

  • Ensure proper sample preparation preserving Lck integrity

  • Check that the transfer method effectively transferred proteins to the membrane

• High background:

  • Optimize blocking conditions (try 5% BSA or commercial blocking buffers)

  • Increase washing frequency and duration

  • Dilute primary and secondary antibodies further

  • Prepare fresh buffers to eliminate contamination

  • Consider using more specific secondary antibodies or detection systems

• Specificity concerns:

  • Validate with Lck-deficient controls

  • Perform peptide competition assays using the immunogen peptide

  • Compare results with a second anti-Lck antibody targeting a different epitope

  • Use phosphatase treatment to confirm phosphorylation-specific signals

• Variable results between experiments:

  • Standardize lysate preparation methodology

  • Maintain consistent antibody handling practices

  • Use internal reference samples across experiments

  • Quantify results relative to loading controls or total protein

  • Consider preparing larger batches of lysates for longitudinal studies

• Cross-reactivity with other SFK members:

  • Include appropriate controls expressing other SFK family members

  • Use more stringent washing conditions

  • Consider immunoprecipitation to isolate Lck specifically before detection

Implementing these troubleshooting strategies will help ensure reliable and reproducible results when using the LCK (Ab-394) Antibody in research applications.

How can LCK (Ab-394) Antibody be used in combination with other antibodies for multiplexed analysis?

The LCK (Ab-394) Antibody can be strategically combined with other antibodies for comprehensive multiplexed analysis of T cell signaling networks. This approach provides richer datasets and reveals relationships between Lck and other signaling components:

• Multi-color Western blotting:

  • Combine LCK (Ab-394) Antibody with phospho-specific antibodies (anti-pY416, anti-pY505)

  • Use antibodies from different host species or directly conjugated antibodies

  • Employ fluorescently-labeled secondary antibodies for simultaneous detection

  • This approach can visualize multiple Lck phosphorylation states in a single sample

• Sequential immunoprecipitation strategies:

  • First immunoprecipitate with phospho-specific antibodies

  • Then detect with LCK (Ab-394) Antibody to quantify specific Lck forms

  • This approach helped identify the double-phosphorylated form (DPho-Lck) of Lck in T cells

• Immunofluorescence co-localization:

  • Combine LCK (Ab-394) Antibody with antibodies against T cell receptor components

  • Use confocal microscopy to analyze spatial relationships

  • Co-localization of anti-pY505 and anti-pY416 staining confirmed DPho-Lck expression in intact T cells

• Flow cytometry panels:

  • If validated for flow cytometry, combine with surface markers and other intracellular signaling proteins

  • This enables correlation of Lck expression with T cell subsets and activation states

• Mass cytometry (CyTOF):

  • Metal-conjugated antibodies enable highly multiplexed analysis

  • Can simultaneously examine dozens of parameters including Lck and its signaling partners

When designing multiplexed analyses, careful antibody selection and validation are essential to avoid cross-reactivity and ensure accurate signal attribution. Appropriate controls for each antibody in the panel must be included.

What are emerging applications for the LCK (Ab-394) Antibody in therapeutic research?

Recent advances in targeted protein degradation technologies are opening new applications for antibodies like LCK (Ab-394) in therapeutic research contexts:

• Validation of Lck-targeting therapeutics:

  • LCK (Ab-394) Antibody can monitor Lck protein levels following treatment with proteolysis-targeting chimeras (PROTACs)

  • Recent research has developed dasatinib-based PROTACs that induce LCK degradation in T-ALL cells

  • The antibody enables quantification of degradation efficiency and selectivity

• Biomarker development:

  • Measuring Lck levels and activation states may serve as biomarkers for:

    • T cell malignancies like T-ALL

    • Autoimmune disease activity

    • Immunotherapy response prediction

  • LCK (Ab-394) Antibody can be used in research developing such diagnostic applications

• Monitoring Lck-targeted therapies:

  • Dasatinib-derived PROTACs broadly target ABL and SRC family kinases including Lck

  • The antibody can help distinguish between inhibition of kinase activity versus protein degradation

  • This information is crucial for understanding drug mechanisms and optimizing therapeutic strategies

• Studying resistance mechanisms:

  • In cases where Lck-targeted therapies fail, the antibody can help investigate whether:

    • Lck protein remains but in an altered conformation

    • Lck is degraded but compensatory pathways are activated

    • Post-translational modifications affect drug binding

• Drug screening platforms:

  • High-throughput screening for compounds affecting Lck expression or stability

  • The antibody provides a reliable readout for such screening efforts

As therapeutic targeting of kinases evolves from inhibition to degradation strategies, antibodies like LCK (Ab-394) play an increasingly important role in monitoring drug effects at the protein level rather than just at the activity level.

How does the double phosphorylated form of Lck (DPho-Lck) impact experimental interpretation?

The discovery of the double phosphorylated form of Lck (DPho-Lck), phosphorylated at both Y394 and Y505, introduces important considerations for experimental design and data interpretation when using the LCK (Ab-394) Antibody:

• Conformational states and antibody accessibility:

  • DPho-Lck exists in an open conformation with pY505 disengaged from the SH2 domain

  • This makes pY505 readily accessible to antibodies in native conditions

  • In contrast, the inactive-closed form (Y394-pY505) has pY505 inaccessible until after denaturation

  • These accessibility differences impact immunoprecipitation and immunofluorescence experiments

• Functional implications:

  • DPho-Lck represents a significant proportion of Lck in T cells (21% in human CD4+ T cells, 29% in Jurkat cells)

  • Despite phosphorylation at the inhibitory Y505 site, this form retains catalytic activity

  • This challenges the binary model of Lck activation/inactivation

• Experimental strategies for identification:

  • Sequential immunoprecipitation with anti-pY416 followed by detection with anti-pY505 (or vice versa)

  • Comparison of immunoprecipitation efficiency before and after denaturation

  • Co-localization analysis using immunofluorescence

• Quantification considerations:

  • Standard Western blotting with phospho-specific antibodies alone cannot distinguish DPho-Lck

  • More sophisticated approaches combining immunodepletion and/or mass spectrometry are required

  • The LCK (Ab-394) Antibody can be used in such approaches to quantify total Lck

Understanding the existence and properties of DPho-Lck is essential for correctly interpreting experimental results and developing a more nuanced view of Lck regulation in T cell biology. This has significant implications for therapeutic strategies targeting Lck in various pathological conditions.

What are the current limitations of LCK (Ab-394) Antibody and how might they be addressed?

While the LCK (Ab-394) Antibody is a valuable research tool, understanding its limitations is important for experimental design and result interpretation:

• Application restrictions:

  • Primary validation is for Western blotting only

  • Researchers must perform their own validation for other applications like immunofluorescence, immunohistochemistry, or flow cytometry

  • Development of application-specific protocols may extend utility

• Specificity considerations:

  • As a polyclonal antibody, batch-to-batch variation may occur

  • Cross-reactivity with closely related Src family kinases should be systematically evaluated

  • Monoclonal antibody alternatives might provide more consistent specificity

• Phosphorylation state detection:

  • Does not specifically distinguish between phosphorylated and non-phosphorylated forms

  • Must be used in combination with phospho-specific antibodies for activation studies

  • Development of conformational-specific antibodies would advance the field

• Quantification challenges:

  • Relative quantification requires careful normalization

  • Absolute quantification requires known standards

  • Mass spectrometry approaches provide complementary quantitative data

Future developments may include:

• Conjugated versions for direct detection in flow cytometry or imaging

• Validation across broader application spectrum

• Integration with emerging proteomics and single-cell analysis technologies

How is research on LCK and related antibodies evolving to address questions in immunotherapy and T cell biology?

Research using tools like the LCK (Ab-394) Antibody is rapidly evolving to address frontier questions in immunotherapy and T cell biology:

• Targeting Lck in T cell malignancies:

  • Beyond kinase inhibition, proteolytic targeting of LCK shows promise for T-ALL treatment

  • PROTAC molecules demonstrate improved exposure-to-efficacy relationships compared to traditional inhibitors

  • LCK (Ab-394) Antibody enables monitoring of degradation efficiency

• Single-cell analysis of Lck activation states:

  • Emerging technologies allow investigation of Lck phosphorylation heterogeneity at single-cell level

  • This may reveal previously unrecognized T cell functional subsets defined by Lck activation patterns

  • Integration with transcriptomic data can link signaling states to gene expression profiles

• Spatiotemporal dynamics of Lck activation:

  • Advanced imaging techniques are revealing how Lck activation spreads within T cell membrane microdomains

  • Understanding these dynamics may inform more precise therapeutic targeting

  • Antibodies with different epitope specificities contribute complementary spatial information

• Role in immunotherapy response and resistance:

  • Lck activation patterns may predict response to checkpoint inhibitors

  • Alterations in Lck phosphorylation could contribute to immunotherapy resistance mechanisms

  • Monitoring Lck as a biomarker might help stratify patients for appropriate immunotherapy approaches

• Structural biology integration:

  • Combining antibody-based detection with structural insights from cryo-EM and X-ray crystallography

  • This integration enhances understanding of how phosphorylation affects Lck conformation and function

  • May guide structure-based drug design for more specific Lck modulators