LGMN Antibody, FITC conjugated

What is LGMN and why is it a significant target for antibody development?

LGMN (Legumain), also known as asparaginyl endopeptidase or AEP, is a lysosomal cysteine endopeptidase belonging to peptidase family C13. It specifically hydrolyzes substrate asparaginyl bonds and has been demonstrated to regulate diverse physiological and pathological processes by remodeling tissue-specific targets . Recent research has established LGMN's significance as it is overexpressed in various tumors including breast, prostatic, and liver cancers, as well as in macrophages composing the tumor microenvironment . This overexpression pattern makes LGMN a pivotal protein in tumor development, invasion, and dissemination, thus becoming an important target for antibody development and cancer research applications .

What are the key molecular characteristics of LGMN that researchers should be aware of?

Researchers working with LGMN should understand several key molecular characteristics:

• LGMN is secreted as inactive prolegumain (56 kDa) and subsequently processed into enzymatically active 46 kDa and 36 kDa forms, as well as a 17 kDa enzymatically inactive C-terminal fragment

• The observed molecular weight in laboratory conditions is typically around 36 kDa

• LGMN has strict specificity for hydrolysis of asparaginyl bonds, though it can also cleave aspartyl bonds slowly, especially under acidic conditions

• It is primarily distributed in lysosomes where it mediates the processing of various albumins, including the conversion of cysteine cathepsin from single-stranded to double-stranded form

• LGMN plays roles in mammalian immunity, particularly in B cells and dendritic cells which function as antigen-presenting cells

Understanding these characteristics is essential when designing experiments and interpreting results using LGMN antibodies.

How does FITC conjugation affect the properties and applications of LGMN antibodies?

FITC (Fluorescein isothiocyanate) conjugation provides LGMN antibodies with fluorescent properties, enabling direct visualization in various imaging applications. FITC-conjugated LGMN antibodies typically have:

• Excitation/emission maxima wavelengths around 493 nm / 522 nm

• Compatibility with standard fluorescence microscopy filters

• Direct detection capability without requiring secondary antibodies

• Applications in immunofluorescence (IF), immunocytochemistry (ICC), immunohistochemistry (IHC), and flow cytometry (FACS)

What are the recommended applications and dilutions for LGMN antibody, FITC conjugated?

Based on the search results, LGMN antibody with FITC conjugation is validated for multiple applications with recommended dilutions varying by application:

Researchers should note that these dilutions are guidelines and should be optimized for specific experimental conditions. As stated in the product information, "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" and dilution requirements may be "sample-dependent" .

What experimental controls should be included when using LGMN antibody, FITC conjugated in immunofluorescence studies?

When conducting immunofluorescence studies with FITC-conjugated LGMN antibody, researchers should include several essential controls:

• Negative control: Samples incubated with isotype-matched IgG (e.g., Mouse IgG1 for CL488-67017 or Rabbit IgG for other products ) at the same concentration as the primary antibody to assess non-specific binding

• Blocking control: Pre-incubation of the antibody with its specific immunogen (where available) to confirm specificity

• Positive control: Tissues known to express LGMN, such as human kidney tissue which has been validated for CL488-67017

• Autofluorescence control: Unstained sample to assess intrinsic tissue/cell fluorescence in the FITC channel

• Cross-reactivity control: If studying multiple species, include samples from each species to verify reactivity, as different LGMN antibodies show varying species reactivity (e.g., some are reactive with human only , while others react with human, mouse, and rat )

These controls ensure reliable interpretation of results and help troubleshoot experimental issues.

How should researchers prepare samples for immunofluorescence staining with LGMN antibody, FITC conjugated?

For optimal results with FITC-conjugated LGMN antibody in immunofluorescence applications, follow these methodological steps:

• Fixation: Use 4% paraformaldehyde or formalin fixation for cells or tissues, typically for 10-20 minutes at room temperature for cells or overnight for tissues

• Permeabilization: After fixation, permeabilize with 0.1-0.5% Triton X-100 in PBS for 10 minutes at room temperature to allow antibody access to intracellular targets

• Blocking: Block with 1-5% BSA (bovine serum albumin) or 5-10% normal serum from the same species as the secondary antibody (if used) for 30-60 minutes to reduce non-specific binding

• Antigen retrieval (for tissue sections): Use appropriate antigen retrieval methods (typically heat-induced epitope retrieval in citrate buffer pH 6.0) if staining paraffin-embedded tissue sections

• Antibody dilution: Prepare the FITC-conjugated LGMN antibody at the recommended dilution (1:50-1:500 for IF-P ) in blocking buffer

• Incubation: Apply diluted antibody to samples and incubate overnight at 4°C or for 1-2 hours at room temperature

• Washing: Wash thoroughly with PBS to remove unbound antibody

• Nuclear counterstain: Apply DAPI or similar nuclear counterstain if desired

• Mounting: Mount with anti-fade mounting medium to preserve fluorescence

• Storage: Store slides in the dark at 4°C to prevent photobleaching until imaging

This protocol may need optimization depending on specific sample types and experimental questions.

What are common issues with weak or non-specific staining when using LGMN antibody, FITC conjugated, and how can they be resolved?

When encountering weak or non-specific staining with FITC-conjugated LGMN antibody, consider these problem-solving approaches:

For weak or no signal:

• Antibody concentration: Increase antibody concentration within the recommended range (e.g., try 1:50 instead of 1:500)

• Antigen masking: Ensure appropriate antigen retrieval is performed for fixed tissues

• Antibody integrity: Verify antibody has not been exposed to repeated freeze-thaw cycles or prolonged light exposure

• Expression levels: Confirm LGMN expression in your sample type; LGMN expression varies by tissue and disease state

• Incubation conditions: Extend incubation time or adjust temperature

For high background or non-specific staining:

• Blocking: Increase blocking time or concentration of blocking agent

• Antibody dilution: Use more diluted antibody solution

• Washing: Perform more stringent washing steps

• Autofluorescence: Use autofluorescence quenching reagents, especially for tissues with high intrinsic fluorescence

• Secondary antibody cross-reactivity: If using additional detection systems, ensure no cross-reactivity with sample components

Each of these adjustments should be tested systematically while maintaining appropriate controls to determine the optimal conditions for your specific experimental setup.

How should LGMN antibody, FITC conjugated be stored to maintain optimal performance?

Proper storage is crucial for maintaining the functionality of FITC-conjugated LGMN antibodies. Based on the search results, follow these storage guidelines:

• Temperature: Store at -20°C for long-term storage . Some products may allow storage at 2-8°C, but freezing is generally recommended for extended preservation

• Light protection: FITC is light-sensitive, so store in amber vials or wrapped in aluminum foil to protect from light exposure

• Aliquoting: Aliquot upon receipt to avoid repeated freeze-thaw cycles; aliquoting is unnecessary for -20°C storage for some products

• Buffer conditions: Most FITC-conjugated LGMN antibodies are supplied in specialized buffers that maintain stability, such as "PBS with 50% Glycerol, 0.05% Proclin300, 0.5% BSA, pH 7.3" or "antibody stabilization buffer"

• Reconstitution: For lyophilized formats (such as those from AssayPro), reconstitute according to manufacturer's instructions just before use

• Expiration: Monitor the expiration date and regularly check for signs of degradation like precipitation or color changes

Following these storage recommendations will help ensure reproducible results across experiments and maximize the useful life of the antibody reagent.

How can LGMN antibody, FITC conjugated be used to investigate the role of LGMN in cancer progression pathways?

LGMN antibody with FITC conjugation provides powerful tools for visualizing LGMN's role in cancer progression. Advanced research applications include:

• Co-localization studies: Using FITC-conjugated LGMN antibodies with other fluorescently-labeled markers to investigate LGMN's interaction with signaling molecules in tumor development pathways, such as:

  • LGMN/PI3K/AKT pathway in breast cancer progression

  • LGMN/TGF-β pathway in tumor-associated macrophage repolarization

  • LGMN/MMP-2 pathway in breast cancer invasion

• Tissue microarray analysis: Studying LGMN expression patterns across multiple tumor samples to correlate with malignancy grades, as higher LGMN expression has been associated with higher tumor grades in breast cancer and increased malignancy in ovarian tumors

• Live-cell imaging: Monitoring LGMN trafficking and localization in living cancer cells to understand its dynamic role in tumor microenvironments

• Flow cytometry: Quantifying LGMN expression levels in different cell populations within heterogeneous tumor samples

• Tumor microenvironment studies: Investigating LGMN expression in both tumor cells and tumor-associated macrophages to understand its dual role in cancer progression

These applications can provide insights into how LGMN contributes to cancer pathology and potentially identify new therapeutic targets or diagnostic markers.

What methodological considerations are important when using LGMN antibody, FITC conjugated in multiparameter flow cytometry?

When incorporating FITC-conjugated LGMN antibody into multiparameter flow cytometry experiments, researchers should consider these methodological details:

• Spectral overlap: FITC (excitation/emission: 493/522 nm ) may have spectral overlap with other fluorophores like PE or GFP. Proper compensation controls must be included:

  • Single-stained controls for each fluorophore

  • Fluorescence minus one (FMO) controls

  • Isotype controls for each antibody class and fluorophore combination

• Sample preparation optimization:

  • For intracellular LGMN detection, use permeabilization reagents compatible with maintaining cell surface marker integrity

  • Test fixation methods that preserve FITC fluorescence while allowing detection of other markers

  • Consider cell-surface vs. intracellular localization of LGMN in different cell states

• Titration: Determine optimal antibody concentration through titration experiments to achieve maximum separation between positive and negative populations while minimizing background

• Panel design:

  • Place LGMN-FITC on a detector with appropriate sensitivity for your expected expression level

  • Consider brightness of FITC when assigning markers to channels (reserve brighter fluorophores for lower-expressed targets)

  • Avoid using FITC for detecting markers in cells with high autofluorescence in the FITC channel

• Analysis strategies:

  • Use appropriate gating strategies that account for shifts in autofluorescence

  • Consider viability dyes to exclude dead cells that may bind antibodies non-specifically

  • For cell populations with varying LGMN expression, use density plots rather than histograms

These considerations will help ensure reliable and reproducible results when using FITC-conjugated LGMN antibody in complex flow cytometry panels.

How should researchers interpret different patterns of LGMN staining in tissue samples?

When analyzing LGMN staining patterns using FITC-conjugated antibodies, researchers should consider these interpretation guidelines:

• Subcellular localization:

  • Lysosomal pattern: Punctate cytoplasmic staining is expected as LGMN is primarily distributed in lysosomes

  • Non-lysosomal pattern: Diffuse cytoplasmic or nuclear staining may indicate alternative forms or functions of LGMN or potential non-specific binding

• Expression intensity correlation:

  • Higher LGMN expression correlates with higher tumor grades in breast cancer

  • Expression intensity should be quantified objectively using appropriate image analysis software when making comparative assessments

• Cell-type specific patterns:

  • LGMN is expressed in both tumor cells and tumor-associated macrophages

  • In immune tissues, expression in B cells and dendritic cells is significant for antigen-presenting functions

  • Different staining patterns between cell types may reflect functional differences

• Processing forms:

  • Different antibodies may recognize different processed forms of LGMN

  • The 36 kDa active form is commonly observed

  • Differences in staining may reflect variations in processing rather than total LGMN levels

• Control comparisons:

  • Always compare staining to appropriate positive and negative controls

  • Human kidney tissue serves as a positive control for some LGMN antibodies

When publishing or presenting LGMN staining results, researchers should include representative images showing the described patterns along with quantification methods and statistical analyses where appropriate.

What quantitative methods are recommended for analyzing LGMN expression data from immunofluorescence studies?

For rigorous quantitative analysis of LGMN expression using FITC-conjugated antibodies in immunofluorescence studies, researchers should employ these methodological approaches:

• Image acquisition standardization:

  • Use consistent exposure settings across all samples and controls

  • Acquire images at the same magnification and resolution

  • Capture multiple fields (minimum 5-10) per sample for statistical validity

  • Include calibration standards when possible

• Signal intensity quantification:

  • Measure mean fluorescence intensity (MFI) within defined regions of interest

  • Use integrated density measurements for total protein expression

  • Apply background subtraction using adjacent negative areas

  • Consider nuclear counterstain for cell count normalization

• Distribution analysis:

  • Quantify subcellular distribution patterns using line-scan analysis

  • Measure co-localization with organelle markers using Pearson's or Mander's coefficients

  • Analyze nuclear/cytoplasmic ratios if relevant

• Software tools:

  • ImageJ/FIJI with appropriate plugins for immunofluorescence analysis

  • CellProfiler for automated multi-parameter cellular analysis

  • Commercial packages like Imaris or ZEN for 3D analysis if using confocal microscopy

• Statistical approaches:

  • Use appropriate statistical tests based on data distribution

  • Report both biological and technical replicates

  • Consider hierarchical analysis for nested experimental designs

  • Use power analysis to determine adequate sample sizes

• Presentation standards:

  • Present data as box plots or violin plots rather than simple bar graphs to show distribution

  • Include representative images alongside quantification

  • Clearly indicate scale bars and imaging parameters

These quantitative approaches ensure reproducible and statistically sound analysis of LGMN expression data, enabling meaningful comparisons across experimental conditions or sample types.

How can researchers validate the specificity of LGMN antibody, FITC conjugated for their particular experimental system?

Validating antibody specificity is crucial for reliable research outcomes. For FITC-conjugated LGMN antibodies, consider these validation strategies:

• Genetic approaches:

  • Compare staining between wild-type cells and LGMN knockout or knockdown models

  • Use siRNA or CRISPR-Cas9 to reduce LGMN expression and confirm corresponding reduction in antibody signal

  • Overexpress LGMN using expression vectors and confirm increased signal

• Biochemical validation:

  • Perform peptide competition assays using the immunogen peptide (e.g., synthetic peptide from amino acid region 100-150 on human Legumain protein )

  • Compare results with alternative LGMN antibodies recognizing different epitopes

  • Correlate LGMN protein detection with mRNA expression data

• Cross-reactivity assessment:

  • Test antibody on samples from different species according to expected reactivity

  • Some LGMN antibodies react with human, mouse, and rat , while others are human-specific

  • Confirm expected molecular weight detection (36 kDa is commonly observed )

• Application-specific validation:

  • For immunofluorescence, compare patterns with published localization data

  • For flow cytometry, include appropriate compensation and FMO controls

  • For western blotting, confirm single band at expected molecular weight

• Independent method correlation:

  • Correlate protein detection with RT-PCR or RNA-seq data

  • Compare with mass spectrometry protein identification when possible

Thorough validation ensures that experimental findings reflect true LGMN biology rather than artifacts of non-specific antibody binding.

What are the advantages and limitations of using LGMN antibody, FITC conjugated compared to other detection methods for studying LGMN in disease models?

Understanding the comparative advantages and limitations of FITC-conjugated LGMN antibodies helps researchers select appropriate methods for their specific research questions:

Advantages:

• Direct visualization: FITC conjugation enables immediate visualization without secondary antibody steps, reducing protocol complexity and potential cross-reactivity issues

• Multiplexing capability: Can be combined with other non-overlapping fluorophores for co-localization studies of multiple targets

• Quantitative potential: Allows quantification of LGMN expression levels through fluorescence intensity measurements

• Spatial information: Provides subcellular localization data not available from methods like western blotting

• Single-cell resolution: Enables analysis of LGMN expression heterogeneity within tissues or cell populations

Limitations:

• Photobleaching: FITC is prone to photobleaching, potentially limiting long-term imaging or requiring special anti-fade mounting media

• Autofluorescence: Tissue autofluorescence in the FITC channel (especially in formalin-fixed tissues) can complicate analysis

• Fixation sensitivity: Some epitopes may be masked by fixation procedures required for immunofluorescence

• Limited sensitivity: May not detect very low LGMN expression levels compared to amplification-based methods

• Form specificity: Antibodies may recognize specific forms of LGMN but not others, potentially missing relevant biological signals

Comparison with alternative methods:

Researchers should select methods based on their specific research questions, considering these comparative strengths and limitations.