LANCL2 Antibody

What is LANCL2 and why is it important for research?

LANCL2 (Lanthionine synthetase C-like protein 2) is a member of the eukaryotic LanC-like protein family, which are homologues of prokaryotic LanC cyclases. LANCL2 functions as a crucial regulator of the serine/threonine protein kinase Akt, particularly in liver cells. It directly facilitates mTORC2 phosphorylation of Akt, promoting maximum Akt activation and cell survival . Research has shown that LANCL2 is widely expressed in specialized organs of the immune system, including blood, spleen, lymph node, and thymus, and is found in various cell types such as T cells, macrophages, endothelial cells, epithelial cells, and dendritic cells, suggesting its potential as a target for immunoregulation . The protein's involvement in multiple signaling pathways and immune responses makes it a valuable target for studies related to cellular homeostasis, immune regulation, and potential therapeutic interventions for autoimmune diseases.

What are the best applications for LANCL2 antibodies in research?

LANCL2 antibodies are versatile tools in research with several key applications. Western blotting is a primary application, as demonstrated in studies where anti-LANCL2 antibodies successfully detected changes in LANCL2 expression levels following knockdown or overexpression experiments . Immunoprecipitation is another critical application, particularly valuable for studying protein-protein interactions, such as the interaction between LANCL2 and Akt or mTORC2 . Immunohistochemistry and immunofluorescence with LANCL2 antibodies enable researchers to examine tissue and cellular localization patterns, which is essential for understanding its distribution across different cell types and tissues including the immune system organs where LANCL2 is prominently expressed . Flow cytometry applications are particularly relevant for immunological research, allowing quantification of LANCL2 expression in specific immune cell populations such as CX3CR1+ macrophages and CD8+ T cells that produce IL-10 . Additionally, LANCL2 antibodies can be employed in chromatin immunoprecipitation (ChIP) assays to investigate potential roles in transcriptional regulation, as LANCL2 has been shown to influence the expression of transcriptional regulators such as NCOR2, Fbxo7, and FOXP1 .

How should LANCL2 antibody specificity be validated?

Validating LANCL2 antibody specificity requires a multi-faceted approach to ensure reliable research outcomes. Researchers should first implement genetic controls by comparing antibody signal between wild-type samples and those with LANCL2 knockdown (using shRNA as demonstrated in the literature) or knockout (such as the LANCL2-/- or LANCL2fl/fl LysCre+ mouse models) . Western blot analysis should show a single band at the expected molecular weight (approximately 50-55 kDa for human LANCL2), with this band disappearing or significantly reducing in LANCL2 knockdown/knockout samples . Peptide competition assays provide another validation method, where pre-incubation of the antibody with a LANCL2-specific peptide should diminish or eliminate signal in subsequent applications. Cross-reactivity testing against other LANCL family members, particularly LANCL1 and LANCL3, is essential due to their homology. Researchers have demonstrated that while LANCL1 is expressed in cells like HepG2, its knockdown does not affect Akt phosphorylation, distinguishing it functionally from LANCL2 . Finally, correlation with orthogonal detection methods should be employed, comparing antibody-based detection with mRNA expression or recombinant protein standards to ensure consistent identification of LANCL2 across multiple experimental platforms .

What are appropriate positive and negative controls for LANCL2 antibody experiments?

For positive controls in LANCL2 antibody experiments, researchers should consider liver cell lines such as HepG2, Hep3B, Ea1C-35, or THLE-2, which have demonstrated consistent LANCL2 expression and functional significance in Akt signaling pathways . Recombinant LANCL2 protein serves as an excellent positive control, particularly when tagged (e.g., with FLAG or His tags) to enable detection with alternative antibodies for verification . Tissues known to express high levels of LANCL2, including spleen, lymph nodes, thymus, and blood-derived immune cells, are also valuable positive controls, especially for immunohistochemistry applications .

For negative controls, LANCL2 knockdown models using shRNA (as demonstrated in HepG2 cells) provide specific cellular negative controls . Genetically modified mouse models lacking LANCL2 (LANCL2-/- or conditional knockouts like LANCL2fl/fl LysCre+ for myeloid-specific deletion) offer tissue-level negative controls . Pre-immune serum or isotype-matched control antibodies should be used as technical negative controls to assess non-specific binding. Additionally, tissues or cells with naturally low LANCL2 expression can serve as biological negative controls, though careful validation is necessary as LANCL2 appears to be widely expressed across multiple tissues and cell types .

What are the optimal conditions for fixation and permeabilization when using LANCL2 antibodies?

Optimal fixation and permeabilization conditions for LANCL2 antibodies depend on the specific application and cellular localization patterns of LANCL2. For immunocytochemistry and immunofluorescence, paraformaldehyde (4%) fixation for 15-20 minutes at room temperature preserves cellular architecture while maintaining antibody epitopes. This is particularly important for LANCL2, which has been shown to interact with both cytoplasmic proteins (Akt) and membrane-associated complexes (mTORC2) . For membrane permeabilization, a gentle approach using 0.1-0.2% Triton X-100 for 5-10 minutes is typically sufficient, as LANCL2 has been found to associate with both cytoplasmic and membrane compartments.

For flow cytometry applications, particularly when studying immune cell populations expressing LANCL2 (such as T cells and macrophages), fixation with 2% paraformaldehyde followed by permeabilization with either 0.1% saponin or commercially available permeabilization buffers designed for intracellular staining is recommended . When performing tissue-based analyses through immunohistochemistry, formalin-fixed paraffin-embedded (FFPE) sections can be used with appropriate antigen retrieval methods, typically heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). For electron microscopy studies of LANCL2 localization, glutaraldehyde fixation with specialized permeabilization protocols may be required to preserve ultrastructural details while allowing antibody access.

How can LANCL2 antibodies be used to investigate LANCL2-Akt-mTORC2 interactions?

Investigating the LANCL2-Akt-mTORC2 interaction axis requires sophisticated antibody-based approaches. Co-immunoprecipitation (Co-IP) with LANCL2 antibodies represents a primary method, as studies have demonstrated that LANCL2 directly interacts with Akt (preferentially inactive Akt) and mTORC2 . Sequential Co-IP experiments can be designed where initial immunoprecipitation with anti-LANCL2 antibodies is followed by a second immunoprecipitation with anti-Akt or anti-rictor (an mTORC2 component) antibodies to isolate the complete complex. The research literature has established that while LANCL2 is not required for Akt-mTORC2 interaction, recombinant LANCL2 enhances Akt phosphorylation by mTORC2 in vitro .

Proximity ligation assays (PLA) offer another powerful approach, using pairs of antibodies against LANCL2 and either Akt or mTORC2 components (such as rictor) to visualize protein interactions in situ with single-molecule resolution. This technique can reveal the subcellular localization of these interactions, which is particularly relevant as Akt activation involves membrane recruitment. For real-time interaction studies, researchers can employ Förster Resonance Energy Transfer (FRET) or Bioluminescence Resonance Energy Transfer (BRET) approaches using fluorescently tagged antibody fragments directed against LANCL2 and its binding partners.

In vitro kinase assays represent another critical application, where immunopurified mTORC2 (using rictor antibodies), purified Akt (as substrate), and recombinant LANCL2 can be combined to assess how LANCL2 facilitates Akt phosphorylation by mTORC2, as demonstrated in published biochemical studies . These methodologies collectively enable a comprehensive analysis of how LANCL2 functions as a molecular scaffold that enhances the efficiency of Akt phosphorylation by mTORC2.

What approaches can be used to study LANCL2 post-translational modifications with antibodies?

Studying LANCL2 post-translational modifications (PTMs) requires specialized antibody-based strategies. Phospho-specific antibodies targeting potential LANCL2 phosphorylation sites represent a primary approach, though these must be developed based on computational prediction of kinase recognition motifs within the LANCL2 sequence. 2D gel electrophoresis followed by western blotting with LANCL2 antibodies can separate different PTM forms based on their charge and mass, revealing the complexity of LANCL2 modifications in different cellular contexts.

Immunoprecipitation with general LANCL2 antibodies followed by mass spectrometry analysis provides a comprehensive PTM mapping strategy, identifying phosphorylation, acetylation, ubiquitination, or other modifications that may regulate LANCL2's interaction with Akt and mTORC2. Subsequent validation of identified PTMs requires either specific antibodies against the modified residues or biochemical approaches.

Chemical treatments that enhance specific PTMs (such as phosphatase inhibitors for phosphorylation or deacetylase inhibitors for acetylation) followed by LANCL2 immunoblotting can reveal which modifications accumulate under different conditions. This is particularly relevant given LANCL2's role in signaling pathways that are regulated by phosphorylation cascades .

Comparing LANCL2 PTM profiles in different tissues or under different stimulation conditions (such as serum or insulin stimulation, which have been shown to activate LANCL2-dependent Akt phosphorylation ) can reveal how modifications contribute to context-specific LANCL2 function. Additional analyses of how PTMs affect LANCL2's ability to enhance mTORC2-mediated Akt phosphorylation would provide mechanistic insights into its regulatory functions.

How do LANCL2 expression patterns differ across immune cell subsets, and what antibody-based methods can detect these differences?

LANCL2 expression varies across immune cell subsets, requiring sophisticated antibody-based detection methods to characterize these differences. Multiparameter flow cytometry represents the gold standard approach, combining LANCL2 antibodies with lineage-specific markers to analyze expression across T cells, B cells, macrophages, dendritic cells, and other immune populations. This approach has revealed that LANCL2 is expressed by T cells, macrophages, endothelial cells, epithelial cells, and dendritic cells , with particularly important functional roles observed in CX3CR1+ macrophages and CD8+ T cells that produce IL-10 .

Single-cell Western blotting technologies enable quantitative analysis of LANCL2 protein levels in individual immune cells, providing insights into population heterogeneity that might be masked in bulk analyses. Mass cytometry (CyTOF) with metal-conjugated LANCL2 antibodies offers high-dimensional phenotyping of immune cell subsets, simultaneously measuring LANCL2 expression alongside dozens of other markers to identify correlations with specific functional states.

Immunohistochemistry and multiplex immunofluorescence of lymphoid tissues provide spatial context to LANCL2 expression patterns, revealing whether certain microenvironmental niches are associated with higher LANCL2 expression. This is particularly relevant given LANCL2's expression in specialized immune organs including spleen, lymph node, thymus, and blood .

Comparative studies between healthy donors and patients with immunological disorders using these techniques can reveal disease-associated alterations in LANCL2 expression. This has relevance for autoimmune conditions like systemic lupus erythematosus (SLE), where LANCL2 has emerged as a therapeutic target with NIM-1324 being developed as an investigational drug . Through these approaches, researchers can comprehensively map LANCL2 expression across the immune system and correlate expression patterns with functional activities in health and disease.

What are the methodological considerations for studying LANCL2 in in vivo disease models?

Studying LANCL2 in disease models requires careful methodological planning. When selecting appropriate animal models, researchers should consider that LANCL2 knockout mice display increased susceptibility in models of infection and inflammation, including H. pylori infection and systemic lupus erythematosus (SLE)-like conditions . Cell-specific conditional knockout models (such as LANCL2fl/fl LysCre+ for myeloid-specific deletion) provide insights into the cell-type-specific contributions of LANCL2 to disease pathogenesis, as demonstrated in H. pylori infection studies where myeloid-specific LANCL2 deletion recapitulated many effects of global LANCL2 knockout .

For tissue collection and processing, standardized protocols that preserve both protein integrity and tissue architecture are essential. This typically involves snap-freezing some samples for molecular analyses while fixing others for histological evaluation with LANCL2 immunostaining. When performing flow cytometry analysis of LANCL2 expression in disease models, optimized tissue dissociation protocols that maintain cellular viability and surface marker expression are critical. This has been successfully implemented in studies examining LANCL2's effects on immune cell populations such as CX3CR1+ macrophages and regulatory T cells in infection and autoimmunity models .

Longitudinal sampling strategies enable tracking of LANCL2 expression and associated immune parameters throughout disease progression. This is particularly relevant for chronic conditions like SLE, where LANCL2-targeting compounds like NIM-1324 have shown therapeutic potential . When testing LANCL2-targeting therapeutics in disease models, appropriate dosing regimens, administration routes, and endpoints must be established to evaluate both efficacy and mechanism of action. Finally, correlation analyses between LANCL2 expression/function and disease parameters provide insights into its pathophysiological significance, as demonstrated by studies correlating macrophage LANCL2 expression with regulatory responses and disease outcomes in infection models .

How can LANCL2 antibodies be used to study its role in phagocyte function and phagosome processing?

LANCL2 antibodies offer multiple approaches to investigate its role in phagocyte function. Immunofluorescence microscopy with LANCL2 antibodies coupled with markers for phagosomal compartments (early endosomes, late endosomes, lysosomes) can reveal the subcellular localization of LANCL2 during phagocytosis and phagosome maturation. This is particularly important given recent findings that loss of LANCL2 in phagocytes impairs phagosome processing, leading to increased uptake of material but decreased markers of endosomal maturation, phagosome turnover, and lysozyme activity .

Live-cell imaging using fluorescently labeled LANCL2 antibody fragments (such as Fab fragments) can track LANCL2 dynamics during active phagocytosis, revealing whether it undergoes redistribution during particle internalization or phagosome maturation. Flow cytometry-based phagocytosis assays combined with LANCL2 staining enable correlation between LANCL2 expression levels and phagocytic capacity across different phagocyte populations or activation states.

Biochemical fractionation of phagosomes at different maturation stages followed by Western blotting for LANCL2 can determine its association with specific phagosomal compartments. This approach can be complemented with proteomic analysis of LANCL2-interacting partners in phagosomes to identify molecular mechanisms underlying its effects on phagosome processing. LANCL2 antibodies can also be used to analyze how pharmacological activation of LANCL2 (e.g., with compounds like NIM-1324) affects phagosome markers and function, as research has shown that such activation increases metabolic and lysozyme activity in the phagosome .

Finally, comparative studies between wild-type and LANCL2-deficient phagocytes using these antibody-based approaches can definitively establish LANCL2's role in phagocytosis and microbial clearance. This has significant implications for understanding how LANCL2 contributes to immune homeostasis and response to infection.

What are common issues with LANCL2 antibody detection, and how can they be resolved?

Researchers commonly encounter several challenges when working with LANCL2 antibodies. Weak signal intensity may occur due to low LANCL2 expression in certain cell types, which can be addressed by using signal amplification methods such as tyramide signal amplification for immunohistochemistry or higher-sensitivity chemiluminescent substrates for Western blotting. High background signal presents another challenge, potentially arising from non-specific antibody binding, which can be minimized by optimizing blocking conditions (typically 5% BSA or milk in TBS-T), increasing washing stringency, or using more dilute antibody concentrations following careful titration experiments.

Cross-reactivity with other LANCL family members (particularly LANCL1 and LANCL3) may occur due to sequence homology. This can be addressed by validating antibody specificity using samples from LANCL2 knockout models or by confirming that the antibody recognizes a unique epitope within LANCL2 . Variability between antibody lots represents another potential issue that can be mitigated by internal standardization protocols where each new lot is validated against previous lots using consistent positive control samples.

For application-specific challenges, Western blotting may be complicated by LANCL2's post-translational modifications creating multiple bands. Using phosphatase treatment of samples prior to electrophoresis can help determine which bands represent phosphorylated forms. In immunoprecipitation applications, weak pull-down efficiency may occur if the antibody epitope overlaps with protein interaction domains. This can be addressed by testing multiple antibodies recognizing different LANCL2 regions or using tagged recombinant LANCL2 as an alternative approach .

How can researchers optimize LANCL2 antibody-based detection in challenging tissue types?

Optimizing LANCL2 antibody detection in challenging tissues requires strategic methodological adaptations. For tissues with high autofluorescence (like liver, where LANCL2 has functional significance ), researchers should employ autofluorescence quenching methods such as Sudan Black B treatment, specialized commercial quenching solutions, or confocal microscopy with spectral unmixing. Tissues with dense extracellular matrix may present antigen accessibility challenges that can be overcome by extended or dual antigen retrieval protocols combining heat-induced epitope retrieval with enzymatic digestion (e.g., proteinase K or pepsin treatment).

Highly vascular tissues may exhibit non-specific antibody binding to endothelial cells or trapped serum proteins. This can be mitigated by perfusion fixation of animal tissues prior to collection or by including additional blocking steps with normal serum matching the secondary antibody species. For tissues with endogenous biotin (which can interfere with biotin-streptavidin detection systems), biotin blocking kits should be used, or alternative detection systems without biotin dependency should be selected.

When working with archival or formalin-fixed paraffin-embedded (FFPE) tissues where epitope masking is common, optimization of antigen retrieval conditions is essential. Systematic testing of different buffers (citrate pH 6.0, EDTA pH 9.0, Tris-EDTA), retrieval durations, and heat sources (microwave, pressure cooker, water bath) can identify optimal conditions for LANCL2 epitope exposure. Finally, for multiplex imaging of LANCL2 alongside other markers in the same tissue section, tyramide signal amplification methods enable sequential antibody labeling and stripping cycles without cross-reactivity, allowing comprehensive characterization of LANCL2's relationships with other proteins in their native tissue context.

What strategies can address contradictory LANCL2 expression data across different antibodies?

When faced with contradictory LANCL2 expression data from different antibodies, researchers should implement systematic validation strategies. Epitope mapping analysis is the first critical step, determining precisely which regions of LANCL2 are recognized by each antibody. Antibodies targeting different epitopes may yield different results due to epitope masking by protein interactions, conformational changes, or post-translational modifications. This is particularly relevant for LANCL2, which has been shown to interact with multiple proteins including Akt and mTORC2 components .

Cross-validation with orthogonal techniques provides another essential approach. Comparing antibody-based protein detection with mRNA expression analysis (qPCR or RNA-seq) can help resolve discrepancies, though remembering that mRNA and protein levels may not always correlate perfectly due to post-transcriptional regulation. Genetic validation using LANCL2 knockdown or knockout models represents a definitive approach to antibody validation. Each antibody should show reduced or absent signal in samples from these models, as demonstrated in studies using shRNA against LANCL2 or LANCL2 knockout mice .

For antibodies showing consistent disagreement, the development of custom validation samples can be valuable. This might include creating cell lines expressing epitope-tagged LANCL2 constructs that can be detected with both the disputed antibodies and tag-specific antibodies to determine which LANCL2 antibody correctly identifies the protein. Multi-antibody approaches should be employed for critical experiments, using at least two antibodies targeting different LANCL2 epitopes and reporting results from both. Finally, method-specific optimization may be necessary, as some antibodies work well for certain applications (e.g., Western blotting) but poorly for others (e.g., immunohistochemistry) due to differences in how epitopes are presented in each method.

How can LANCL2 antibodies be used to evaluate therapeutic responses to LANCL2-targeting compounds?

LANCL2 antibodies provide essential tools for evaluating therapeutic responses to LANCL2-targeting compounds such as NIM-1324, which is being developed for systemic lupus erythematosus (SLE) . Target engagement assays represent a primary application, where LANCL2 antibodies can be used in cellular thermal shift assays (CETSA) or drug affinity responsive target stability (DARTS) approaches to demonstrate direct binding of compounds to LANCL2 protein. These methods rely on the principle that ligand binding often alters protein stability, which can be detected through differential antibody recognition following thermal or proteolytic challenges.

Pharmacodynamic biomarker development is another critical application. LANCL2 antibodies can be used to monitor downstream signaling changes following therapeutic LANCL2 targeting, such as alterations in regulatory T cell populations (CD25hiFOXP3+ Tregs), which increase with NIM-1324 treatment while inflammatory IL-17+ and IL-21+ CD4+ T cell subsets decrease . Immunohistochemistry or flow cytometry with LANCL2 antibodies can assess whether treatment with LANCL2-targeting compounds alters LANCL2 expression or cellular distribution in target tissues, potentially revealing feedback mechanisms.

For patient stratification, LANCL2 antibodies could enable screening for baseline LANCL2 expression or localization patterns that might predict treatment response. This is particularly relevant for heterogeneous conditions like SLE, where patient subsets might benefit differently from LANCL2-targeted therapy. Ex vivo testing systems can also be developed, where patient-derived samples are treated with LANCL2-targeting compounds and monitored for relevant biological responses. Research has demonstrated that ex vivo treatment of human PBMCs from SLE patients with NIM-1324 results in reduced levels of IFN-α, IL-6, and IL-8, providing a potential predictive assay for therapeutic efficacy .

What considerations are important when using LANCL2 antibodies in biomarker development for autoimmune diseases?

Developing LANCL2 as a biomarker for autoimmune diseases requires careful methodological consideration. The selection of appropriate LANCL2 antibodies with validated specificity is the foundation of reliable biomarker development. Antibodies should undergo rigorous validation in relevant disease models, including LANCL2 knockout controls and samples from patients with the targeted autoimmune disease . The choice of detection platform significantly impacts biomarker utility, with options including immunohistochemistry for tissue expression patterns, flow cytometry for cellular analysis, ELISA for quantitative measurement in body fluids, or multiplex systems for simultaneous assessment of LANCL2 alongside other immune markers.

Standardization protocols must be established to ensure reproducibility across laboratories and clinical settings. This includes standard operating procedures for sample collection, processing, storage, antibody concentrations, incubation conditions, and data analysis methods. Reference ranges for LANCL2 expression or activation status in healthy individuals must be established, accounting for potential confounding factors such as age, sex, and inflammatory status. This is particularly important given LANCL2's roles in immune regulation and potential as a therapeutic target in conditions like SLE .

Correlation with clinical parameters is essential for biomarker validation, assessing how LANCL2 measurements relate to disease activity scores, specific organ involvement, treatment response, or long-term outcomes. For example, LANCL2 expression changes could be correlated with SLE Disease Activity Index (SLEDAI) scores or specific manifestations like nephritis. Longitudinal sampling strategies enable tracking LANCL2 biomarker dynamics throughout disease progression and treatment, potentially identifying patterns that predict flares or remission.

Additionally, technology transfer considerations for clinical implementation must be addressed, including developing simplified protocols suitable for clinical laboratories and potentially creating companion diagnostic tests for LANCL2-targeting therapeutics like NIM-1324 .