LIME1 Antibody

What is LIME1 and what cellular functions does it serve?

LIME1 (Lck-interacting transmembrane adaptor 1) is a 30 kDa double-palmitoylated transmembrane adaptor protein predominantly expressed in T cells. It plays a critical role in T-cell receptor (TCR) signaling by linking receptor stimulation to downstream signaling pathways. Recent research demonstrates that LIME1 is essential for inflammatory chemokine-mediated signaling and the subsequent migration of effector T cells to inflammatory sites. In LIME1-knockout mice, T cell-mediated inflammatory responses are significantly impaired due to reduced migration and defective morphological polarization in response to inflammatory chemokines such as CCL5 and CXCL10 . While LIME1 primarily serves as a positive regulator of TCR-dependent signaling by associating with Lck and Fyn kinases, under certain conditions it may mediate inhibitory signals .

How does LIME1 function in immune cell signaling pathways?

LIME1 functions as a critical transmembrane adaptor in T cell signaling cascades. Upon T cell receptor stimulation, LIME1 is phosphorylated by Lck, a Src-family kinase, which creates binding sites for downstream signaling molecules. Unlike LAT (Linker for Activation of T cells), which is phosphorylated mainly by ZAP-70 or Syk, LIME1 interacts with and is phosphorylated by Lck. This phosphorylation enables LIME1 to recruit cytoplasmic proteins including Lck, Fyn, p85PI3K, Grb2, Gads, Shp-2, Vav, and Csk to membrane-proximal sites . When overexpressed in Jurkat T cells, LIME1 enhances TCR-mediated signaling pathways driving IL-2 production . Additionally, LIME1 plays a specific role in regulating chemokine-mediated T cell migration, particularly affecting the activation of small GTPases Rac1 and Rap1 and the phosphorylation of Pyk2 and Cas signaling proteins .

What should I consider when choosing between different LIME1 antibody clones?

When selecting LIME1 antibody clones, researchers should consider several critical factors:

• Epitope specificity: Different clones target distinct regions of LIME1. For example, LIME-06 recognizes amino acids 141-295 , LIME-10 targets amino acids 281-296 , and mLIME-05 binds to amino acids 86-202 . The epitope location may affect recognition of specific LIME1 isoforms or detection of the protein in certain experimental conditions.

• Host species and isotype: Available options include mouse monoclonal (IgG1, IgG2a), rabbit polyclonal, and goat polyclonal antibodies . Choose based on compatibility with your secondary detection system and other antibodies in multiplexing experiments.

• Validated applications: Antibody clones have varying performance across applications. For instance, LIME-10 is validated for Western blotting and immunohistochemistry (paraffin) , while LIME-06 is optimized for immunoprecipitation and flow cytometry .

• Species reactivity: Some antibodies recognize human LIME1 (LIME-06, LIME-10), while others detect mouse LIME1 (mLIME-05) or both (antibodies with cross-reactivity to mouse and rat) .

Selection should align with your specific experimental needs and the biological context of your research question.

What are the advantages of monoclonal versus polyclonal LIME1 antibodies?

Monoclonal and polyclonal LIME1 antibodies offer distinct advantages for different research applications:

Monoclonal Antibodies:

• Provide superior lot-to-lot consistency, ensuring reproducible results across experiments and time

• Offer high specificity for particular epitopes, such as the LIME-10 clone recognizing the C-terminal region (aa 281-296)

• Ideal for applications requiring precise epitope targeting, like mapping post-translational modifications

• Available options include mouse monoclonals like LIME-06 (binding aa 141-295) for human samples and mLIME-05 (targeting aa 86-202) for mouse samples

Polyclonal Antibodies:

• Recognize multiple epitopes, potentially providing stronger signal amplification

• May maintain reactivity even if some epitopes are masked by protein interactions or modifications

• Useful for detecting native proteins across diverse experimental conditions

• Examples include rabbit polyclonal antibodies targeting the C-terminal region (aa 190-219) of human LIME1

The choice depends on experimental requirements: use monoclonals when specificity and reproducibility are paramount, and polyclonals when maximum sensitivity and epitope flexibility are needed.

What are the optimal conditions for using LIME1 antibodies in Western Blotting?

For optimal Western blotting detection of LIME1, researchers should consider these protocol recommendations:

• Sample preparation: LIME1 is a 30 kDa transmembrane protein that may require special consideration for extraction. Membrane protein extraction buffers containing mild detergents are recommended to preserve protein integrity .

• Antibody dilutions:

  • For E3M3B rabbit monoclonal: 1:1000 dilution is recommended

  • For LIME-10 mouse monoclonal: 1 μg/ml under reducing conditions

  • For polyclonal antibodies: Starting dilution of 1:1000 is suggested for initial optimization

• Detection system: Since LIME1 has a relatively low molecular weight (30 kDa), high-resolution gels (12-15%) provide better separation. For enhanced sensitivity, some antibodies are available with direct conjugation to HRP or fluorescent tags .

• Positive controls: Jurkat T cell or CEM cell lysates serve as effective positive controls, with documented expression of LIME1 . For mouse studies, lymphatic tissues like spleen or thymus are appropriate .

• Blocking conditions: Standard blocking with 5% non-fat dry milk or BSA in TBST is typically sufficient, though optimization may be required for specific antibodies .

The observed molecular weight should be approximately 30 kDa, though post-translational modifications may affect migration patterns .

How can I validate LIME1 antibody specificity in my experiments?

Validating LIME1 antibody specificity requires a multi-faceted approach:

• Positive and negative control samples: Compare LIME1 expression between cells known to express the protein (T lymphocytes) and those with minimal expression. The antibody should detect a ~30 kDa band in positive samples but show minimal signal in negative samples .

• Knockout/knockdown validation: The gold standard approach is to test antibody reactivity in LIME1 knockout or knockdown samples. LIME1-/- mouse models are available and can serve as definitive negative controls for mouse-reactive antibodies .

• Peptide competition: Pre-incubate the antibody with the immunizing peptide before application to samples. For example, with LIME-10 antibody, the blocking peptide corresponding to aa 281-296 should abolish specific signal .

• Cross-reactivity assessment: Some antibodies may cross-react with related proteins. For instance, antibodies against CD170 may cross-react with Siglec-14, whose first two Ig domains are nearly identical to CD170 . Verify specificity against related transmembrane adaptor proteins.

• Transfection studies: Compare antibody reactivity in cells transfected with LIME1 expression vectors versus mock-transfected controls. This approach is particularly valuable for antibodies like LIME-06, which has been validated using J77-LIME transfectants versus Jurkat cells .

What controls should I use when performing immunoprecipitation with LIME1 antibodies?

For rigorous immunoprecipitation experiments using LIME1 antibodies, the following controls are essential:

• Isotype control: Use a matched isotype antibody (e.g., mouse IgG2a for LIME-10 or mouse IgG1 for LIME-06) from the same host species to assess non-specific binding . This control should undergo identical immunoprecipitation procedures.

• Input sample: Always include an aliquot (5-10%) of the pre-immunoprecipitation lysate to confirm the presence of LIME1 in your starting material and to assess enrichment efficiency.

• Negative control lysate: Process lysates from cells known to express minimal or no LIME1 to identify any non-specific bands.

• Knockout/knockdown control: When available, lysates from LIME1 knockout or knockdown cells provide the most stringent control for specificity .

• Cross-validation: For critical experiments, consider confirming results using a second LIME1 antibody recognizing a different epitope. For example, if primary IP uses LIME-06 (aa 141-295), verification could employ LIME-10 (aa 281-296) .

For optimal results with LIME1 immunoprecipitation, the recommended antibody dilution is 1:50 for the E3M3B rabbit monoclonal antibody . When using mouse monoclonals like mLIME-05, protein A affinity chromatography-purified antibodies with >95% purity (by SDS-PAGE) yield the best results .

How can LIME1 antibodies be used to study T-cell signaling and migration?

LIME1 antibodies offer powerful tools for investigating T-cell signaling pathways and migration mechanisms:

• Phosphorylation dynamics: Using phosphorylation-specific antibodies alongside total LIME1 antibodies enables researchers to track LIME1 activation following T-cell receptor stimulation or chemokine exposure. This approach has revealed that LIME1 is critical for chemokine-mediated activation of Rac1 and Rap1, and for the phosphorylation of Pyk2 and Cas signaling proteins .

• Protein-protein interaction studies: Immunoprecipitation with LIME1 antibodies (such as LIME-06 or mLIME-05) followed by immunoblotting for binding partners like Lck, Fyn, or Csk can map signaling complexes formed under different stimulation conditions . Co-localization studies using fluorescently-tagged LIME1 antibodies provide spatial information about these interactions.

• T-cell migration assays: LIME1 antibodies are valuable tools for investigating the role of this adaptor in chemokine-mediated T-cell migration. Studies using LIME1-deficient models have demonstrated that LIME1 is essential for the migration and morphological polarization of effector T cells in response to inflammatory chemokines like CCL5 and CXCL10 .

• Raft-associated signaling: Since LIME1 is a raft-associated transmembrane adaptor, antibodies targeting this protein can be used in lipid raft isolation protocols to study compartmentalized signaling events following TCR stimulation .

These applications have contributed to our understanding of LIME1 as a critical regulator of inflammatory chemokine-mediated signaling and T-cell migration to inflammatory sites.

What are the considerations for using LIME1 antibodies in flow cytometry?

When employing LIME1 antibodies for flow cytometry analysis, researchers should consider these important technical aspects:

• Permeabilization requirements: As LIME1 is primarily localized in the cytoplasmic domain, effective permeabilization is essential. Standard protocols using commercial intracellular fixation and permeabilization buffer sets (such as those from eBioscience/Thermofisher) are recommended . The membrane domain must be effectively permeabilized to allow antibody access to cytoplasmic epitopes.

• Antibody selection: For flow cytometry, antibodies validated specifically for this application should be selected. The LIME-06 clone (binding aa 141-295) has been validated for flow cytometry (FACS) , while some polyclonal antibodies may also be suitable with appropriate optimization.

• Concentration optimization: The recommended starting concentration is 1-5 μg/ml, though this should be titrated for optimal signal-to-noise ratio with your specific cell types .

• Controls for intracellular staining: Include appropriate isotype controls (matching the host species and isotype of your LIME1 antibody) and use LIME1-negative cells as biological controls. For mouse IgG1 antibodies, the MOPC-21 isotype control is appropriate .

• Fluorochrome selection: When using directly conjugated antibodies, consider the expression level of LIME1 in your cells of interest. For low-abundance proteins, brighter fluorochromes (PE, APC) may be preferable to FITC or other less intense options .

Flow cytometry analysis has confirmed that LIME1 is preferentially expressed in effector T cells, providing important insights into its functional role in adaptive immunity .

What are common issues when using LIME1 antibodies and how can they be resolved?

Researchers frequently encounter these challenges when working with LIME1 antibodies:

• Weak or absent signal in Western blotting:

  • Problem: LIME1's membrane localization can make extraction challenging

  • Solution: Use specialized membrane protein extraction buffers with appropriate detergents; avoid excessive heating which may cause aggregation of transmembrane proteins

  • Alternative: Try antibodies targeting different epitopes—C-terminal antibodies (LIME-10) may perform differently from those targeting internal domains (LIME-06)

• Non-specific bands:

  • Problem: Some antibodies may cross-react with structurally similar proteins

  • Solution: Include appropriate controls such as LIME1 knockout/knockdown samples

  • Alternative: Use higher antibody dilutions (1:2000 instead of 1:1000) to reduce background, or try monoclonal antibodies with higher specificity

• Variability between experiments:

  • Problem: Inconsistent results between replicates

  • Solution: Maintain strict consistency in protocols; for critical applications, consider recombinant antibodies that offer superior lot-to-lot consistency

  • Alternative: Pool results from multiple antibodies targeting different LIME1 epitopes to confirm observations

• Poor immunoprecipitation efficiency:

  • Problem: Low yield in pull-down experiments

  • Solution: Use antibodies specifically validated for IP; the recommended dilution for E3M3B rabbit mAb is 1:50

  • Alternative: Pre-clear lysates thoroughly and ensure antibody concentration is sufficient (typically 1-5 μg antibody per 100-500 μg total protein)

These troubleshooting approaches address the most common technical challenges while maintaining scientific rigor in LIME1 research applications.

How do post-translational modifications affect LIME1 antibody binding?

Post-translational modifications (PTMs) of LIME1 can significantly impact antibody binding and experimental outcomes:

• Phosphorylation effects: LIME1 undergoes tyrosine phosphorylation after TCR stimulation or CD4/CD8 co-receptor ligation. Antibodies targeting regions containing phosphorylation sites may have altered binding affinity to the phosphorylated versus non-phosphorylated forms. This is particularly relevant for antibodies recognizing the cytoplasmic domain, which contains multiple tyrosine residues that serve as docking sites for SH2 domain-containing proteins .

• Palmitoylation considerations: LIME1 is a double-palmitoylated protein, and this lipid modification is crucial for its raft localization. Palmitoylation might affect protein conformation and potentially mask certain epitopes. Antibodies targeting the membrane-proximal region might be particularly affected by this modification .

• Epitope accessibility in protein complexes: Upon activation, LIME1 forms complexes with multiple signaling proteins including Lck, Fyn, p85PI3K, Grb2, and others. These interactions may sterically hinder antibody access to certain epitopes. When studying protein-protein interactions, using antibodies recognizing epitopes distant from known interaction domains may be advantageous .

• Selection strategies for different experimental questions:

  • For detecting total LIME1 regardless of activation state: Choose antibodies targeting regions unlikely to be affected by PTMs, such as the N-terminal or C-terminal domains away from known modification sites

  • For studying activation-dependent events: Consider using phospho-specific antibodies that specifically recognize phosphorylated forms of LIME1

  • For immunoprecipitation of protein complexes: Select antibodies unlikely to disrupt protein-protein interactions of interest

Understanding these considerations is essential for accurate interpretation of experimental results when studying LIME1 dynamics in T-cell signaling.