LGALS1 Recombinant Monoclonal Antibody

What is LGALS1 and why is it an important research target?

LGALS1 (Lectin, Galactoside-Binding, Soluble, 1) is a 14.7 kDa protein also known as Galectin-1, GAL1, or GBP. It functions as a β-galactoside-binding protein involved in cell-cell and cell-matrix interactions. Its significance in research stems from its roles in immune regulation, cancer progression, and neuroinflammation. Understanding LGALS1 function has implications for autoimmune diseases, cancer immunotherapy, and inflammatory conditions, making reliable antibodies against this target crucial for advancing these research areas .

What distinguishes recombinant monoclonal antibodies against LGALS1 from other antibody types?

Recombinant monoclonal antibodies against LGALS1 are generated using recombinant DNA technology, ensuring batch-to-batch consistency and specific epitope recognition. Unlike polyclonal antibodies, which recognize multiple epitopes, recombinant monoclonal antibodies bind to a specific epitope segment (such as amino acids 12-108 of LGALS1), providing higher specificity and reproducibility in experimental results . The recombinant production method also eliminates animal-to-animal variation inherent in traditional hybridoma-derived monoclonal antibodies, making them ideal for longitudinal studies and applications requiring consistent performance.

How should researchers select the appropriate LGALS1 antibody for their specific application?

Selection should be guided by matching the antibody's validated applications to research needs. First, determine which technique will be employed (WB, IHC, IF, ELISA, FCM), then confirm the antibody has been validated for that application. For example, ABIN6939959 is validated for ELISA, IHC, coating applications, and staining methods, while the 8A12 Mouse mAb is validated for Western blotting and immunohistochemistry . Second, verify species reactivity—most LGALS1 antibodies react with human Galectin-1, but cross-reactivity with mouse or rat orthologs varies between products. Finally, consider the specific epitope recognition (e.g., AA 12-108 vs. full-length protein) as this may impact recognition of specific isoforms or post-translationally modified forms of LGALS1.

What are the optimal dilution conditions for LGALS1 recombinant monoclonal antibodies across different applications?

The optimal dilution varies by application and specific antibody clone. Based on product guidelines, for Western blotting, the recommended dilution for many LGALS1 antibodies is 1:1000, as specified for the 8A12 Mouse mAb . For immunohistochemistry (paraffin sections), a 1:200 dilution is typically recommended . ELISA applications generally require less dilute solutions (1:100 to 1:500), but this varies by kit and protocol. For immunofluorescence, dilutions commonly range from 1:100 to 1:500. Flow cytometry applications may require more concentrated antibody solutions. Always perform titration experiments with your specific sample types to determine optimal conditions, as factors such as LGALS1 expression levels and sample preparation methods can influence antibody performance.

How should researchers validate LGALS1 antibody specificity for their experimental system?

A comprehensive validation strategy should include multiple approaches. First, perform Western blotting with positive controls (tissues/cells known to express LGALS1) and negative controls (LGALS1 knockout samples or tissues with minimal expression). The expected molecular weight band of approximately 15 kDa should be observed . Second, use RNA interference (siRNA/shRNA) to knock down LGALS1 in your experimental system and confirm decreased antibody signal. Third, consider using multiple antibodies targeting different epitopes of LGALS1 to ensure consistent results. For immunohistochemistry applications, include peptide blocking experiments where pre-incubation of the antibody with the immunizing peptide should eliminate specific staining. Finally, orthogonal validation using non-antibody techniques (e.g., mass spectrometry or RNA expression) provides additional confidence in antibody specificity.

What sample preparation protocols are recommended for detecting LGALS1 in different cellular compartments?

LGALS1 localization can vary depending on cell type and physiological state, requiring specific sample preparation approaches. For cytoplasmic LGALS1, standard cell lysis buffers containing non-ionic detergents (0.5% NP-40 or 1% Triton X-100) are typically sufficient. For nuclear LGALS1, use nuclear extraction protocols with higher salt concentrations (300-500 mM NaCl) to efficiently extract nuclear proteins. For secreted LGALS1, concentrate culture media using precipitation methods or centrifugal filters before analysis. When studying membrane-associated LGALS1, consider crosslinking protocols before extraction to preserve transient interactions. For immunofluorescence, different fixation methods may reveal different pools of LGALS1: paraformaldehyde (4%) preserves most localizations, while methanol fixation may better reveal nuclear LGALS1. In all cases, include phosphatase and protease inhibitors to prevent degradation during sample preparation.

How can LGALS1 recombinant monoclonal antibodies be utilized in multiplexed imaging studies?

Multiplexed imaging with LGALS1 antibodies requires careful antibody selection and optimization. Begin by selecting LGALS1 recombinant antibodies from different host species (e.g., rabbit or mouse) than your other target antibodies to avoid cross-reactivity . For fluorescence multiplexing, directly conjugated antibodies minimize background and crosstalk, though unconjugated primary antibodies with appropriate secondary antibodies can be used with proper controls. Sequential staining protocols with stripping or quenching steps between rounds can expand multiplexing capacity. For mass cytometry or imaging mass cytometry applications, metal-conjugated anti-LGALS1 antibodies enable detection alongside dozens of other targets. Cyclic immunofluorescence methods allow for 30+ targets on the same tissue section by iterative staining, imaging, and signal removal. In all cases, validate the protocol using single-stained controls to confirm lack of interference between antibodies and preservation of LGALS1 epitopes throughout multiplexed detection workflows.

What approaches can be used to study LGALS1 protein-protein interactions using recombinant monoclonal antibodies?

Several advanced approaches leverage LGALS1 antibodies for protein interaction studies. Co-immunoprecipitation (Co-IP) using LGALS1 recombinant monoclonal antibodies can pull down LGALS1 along with its binding partners for subsequent identification by mass spectrometry or Western blotting. Proximity ligation assays (PLA) combine LGALS1 antibodies with antibodies against suspected interaction partners to generate fluorescent signals only when proteins are within 30-40 nm of each other, providing in situ visualization of interactions. For live-cell applications, conjugate LGALS1 antibodies with FRET (Förster Resonance Energy Transfer) compatible fluorophores to monitor real-time interactions when paired with fluorescently-labeled candidate partners. ChIP-seq (Chromatin Immunoprecipitation Sequencing) can identify LGALS1 interactions with genomic regions if nuclear localization is being studied. Importantly, ensure that the epitope recognized by the LGALS1 antibody (e.g., amino acids 12-108) doesn't overlap with protein interaction domains to avoid interference with the interactions being studied .

How can researchers quantitatively assess LGALS1 expression levels across different tissue or cell types?

Quantitative assessment of LGALS1 requires standardized approaches across samples. For tissue analysis, digital pathology platforms can quantify LGALS1 immunohistochemistry signals using LGALS1 recombinant monoclonal antibodies at standardized dilutions (typically 1:200) , with automated scoring algorithms to measure intensity, percent positive cells, and subcellular localization patterns. Flow cytometry provides single-cell quantification of LGALS1 in cell suspensions, with signal calibration using beads with known antibody binding capacity to convert fluorescence intensity to molecules per cell. For Western blotting quantification, include a standard curve of recombinant LGALS1 protein alongside samples and use chemiluminescence or fluorescence detection within the linear range of the assay. Absolute quantification can be achieved using mass spectrometry with isotope-labeled peptide standards corresponding to LGALS1 sequences. When comparing across tissue types, normalize LGALS1 expression to appropriate housekeeping proteins selected for consistent expression in the tissues being compared.

How should researchers address non-specific binding when using LGALS1 recombinant monoclonal antibodies?

Non-specific binding can be systematically addressed through several optimization strategies. First, increase blocking stringency by extending blocking time (1-2 hours) and using specific blockers tailored to your sample type: 5% BSA for Western blots, 10% serum from the secondary antibody host species for immunohistochemistry, or commercial blockers containing both proteins and detergents. Second, optimize antibody dilution, as too concentrated antibody solutions increase background—perform a dilution series from 1:100 to 1:2000 to identify the optimal signal-to-noise ratio . Third, add 0.1-0.3% Triton X-100 or Tween-20 to washing buffers and increase washing duration and number of washes. For tissues with high endogenous biotin or peroxidase activity, include specific blocking steps before antibody incubation. If background persists, consider switching from ABC-based detection to polymer-based detection systems in IHC applications. Finally, pre-adsorb the antibody with proteins from the species being tested or use isotype controls at the same concentration as your LGALS1 antibody to distinguish specific from non-specific signals.

What factors might cause variability in LGALS1 detection across different experimental replicates?

Multiple factors can contribute to inter-experimental variability. Antibody factors include lot-to-lot variations (less common with recombinant antibodies but still possible), freeze-thaw cycles, and storage conditions—aliquot antibodies upon receipt and store at -20°C or -80°C as recommended . Sample-related variability can stem from inconsistent sample collection timing (LGALS1 expression may fluctuate with cell cycle or activation state), variable fixation times affecting epitope accessibility, or inconsistent lysis conditions leading to different extraction efficiencies. Technical factors include inconsistent transfer efficiency in Western blotting, variable antibody incubation temperatures, and automated vs. manual processing differences. Biological variability can result from heterogeneous LGALS1 expression within cell populations or tissue regions. To minimize variability, standardize all protocols with detailed SOPs, include consistent positive controls across experiments, consider using automated systems where possible, and implement quantitative methods with appropriate normalization to internal controls.

How can researchers differentiate between different isoforms or post-translational modifications of LGALS1 using available antibodies?

Distinguishing LGALS1 variants requires strategic antibody selection and complementary techniques. First, select antibodies recognizing epitopes that span or exclude regions with known modifications—antibodies targeting amino acids 12-108 may detect different forms than those targeting the full-length protein (amino acids 1-135) . For distinguishing isoforms, use high-resolution gel systems (15-20% acrylamide or gradient gels) in Western blotting to separate closely migrating bands. To identify specific post-translational modifications (PTMs), combine immunoprecipitation using LGALS1 antibodies with subsequent probing using modification-specific antibodies (e.g., anti-phospho, anti-acetyl). For comprehensive PTM analysis, immunoprecipitate LGALS1 using validated antibodies and analyze by mass spectrometry. 2D gel electrophoresis (separating by both isoelectric point and molecular weight) followed by Western blotting can resolve modified forms of LGALS1. To confirm the identity of specific bands, include controls such as dephosphorylation treatments or site-directed mutagenesis of predicted modification sites. Additionally, the use of multiple antibodies recognizing different epitopes can provide verification of isoform identity.

How do recombinant monoclonal antibodies against LGALS1 compare to polyclonal antibodies in detecting low-abundance LGALS1 in tissues?

The detection of low-abundance LGALS1 reveals significant performance differences between antibody types. Recombinant monoclonal antibodies offer superior specificity by recognizing single epitopes (e.g., amino acids 12-108 of LGALS1) , which significantly reduces background signal in tissues with low target expression. This specificity enables reliable detection of LGALS1 even in tissues with minimal expression levels. While polyclonal antibodies theoretically provide signal amplification through multiple epitope binding, their variable batch composition introduces inconsistency and potential cross-reactivity with structurally similar proteins. In comparative studies, recombinant monoclonal antibodies typically demonstrate better signal-to-noise ratios in immunohistochemical applications at standard dilutions (1:200) , particularly critical for distinguishing true low-level expression from background. Additionally, the clonal nature of recombinant antibodies ensures consistent performance across experiments, enabling more reliable quantification of subtle expression differences between tissue types or experimental conditions. For maximum sensitivity while maintaining specificity, signal amplification systems like tyramide signal amplification can be paired with recombinant monoclonal antibodies, offering superior detection capabilities compared to standard detection with polyclonal antibodies.

What are the comparative advantages of different application methods when detecting LGALS1 in clinical samples?

Each detection method offers distinct advantages for LGALS1 analysis in clinical contexts. Immunohistochemistry (IHC) using LGALS1 recombinant monoclonal antibodies at 1:200 dilution preserves tissue architecture, allowing correlation of LGALS1 expression with histopathological features and spatial relationships with other markers. IHC works effectively with formalin-fixed paraffin-embedded (FFPE) specimens, making it compatible with standard clinical sample processing. Western blotting provides higher specificity for detecting the 15 kDa LGALS1 protein and can distinguish full-length protein from fragments, but requires tissue disruption and higher sample volumes. Flow cytometry enables simultaneous analysis of LGALS1 with multiple markers at the single-cell level, ideal for heterogeneous samples like blood or disaggregated tumors. ELISA offers quantitative measurement with high sensitivity and throughput for bodily fluids or tissue extracts. For comprehensive clinical analysis, integrating multiple methods provides complementary data: IHC for localization, Western blotting for specificity confirmation, flow cytometry for cellular heterogeneity, and ELISA for absolute quantification. The selection should be guided by the specific clinical question, sample availability, and required sensitivity/specificity balance.

How should researchers compare and interpret data generated using different clones of LGALS1 recombinant monoclonal antibodies?

Comparing data from different LGALS1 antibody clones requires systematic analysis and careful interpretation. First, document the epitope specificity of each clone—some target specific regions (e.g., amino acids 12-108) while others recognize the full-length protein (amino acids 1-135). Epitope differences can affect accessibility in certain applications or under specific conditions. Create a standardized sample set that includes positive controls (high LGALS1 expressors), negative controls (LGALS1-knockdown or knockout samples), and a range of samples with varied expression levels. Process this panel with each antibody clone using identical protocols and analyze in parallel. Quantify relative signals using digital image analysis for immunohistochemistry or densitometry for Western blots, creating antibody-specific calibration curves. When comparing across applications, recognize that optimal dilutions differ by clone and method—for example, the 8A12 clone uses 1:1000 for Western blotting but 1:200 for IHC . For integrating data from multiple studies using different clones, include internal reference standards to establish conversion factors between datasets. Finally, consider that discrepancies between antibody results may reveal biologically significant information about protein conformations, modifications, or interactions that differentially affect epitope accessibility rather than representing technical artifacts.

Table 1: Comparison of LGALS1 Recombinant Monoclonal Antibody Properties and Applications