LGR5 Antibody

What is LGR5 and why is it an important research target?

LGR5 is a G protein-coupled receptor that functions as a receptor for R-spondins, potentiating the canonical Wnt signaling pathway. The canonical LGR5 protein is 907 amino acids in length with a molecular weight of approximately 100 kDa, featuring Golgi and membrane subcellular localization . LGR5 serves as a biomarker for adult stem cells in specific tissues and has gained significant attention due to its overexpression in multiple cancer types, particularly colorectal cancer (CRC), hepatocellular carcinoma (HCC), and pre-B-ALL tumors .

LGR5 functions in G protein-coupled receptor activity and G protein-coupled peptide receptor activity, playing a crucial role in signal transduction . Unlike classical G-protein coupled receptors, LGR5 does not activate heterotrimeric G-proteins to transduce signals but instead associates with phosphorylated LRP6 and frizzled receptors upon binding to R-spondins . This mechanism is central to its role in stem cell maintenance and cancer development.

What types of LGR5 antibodies are available for research applications?

Researchers can access a diverse range of LGR5 antibodies:

• Monoclonal antibodies: Highly specific antibodies like clone OTI2A2 that target defined epitopes

• Polyclonal antibodies: Generated against specific regions of the LGR5 protein

• Domain-specific antibodies: Some targeting the extracellular domain (N-terminal 101 amino acids), which is particularly valuable for live cell applications

• Species-specific antibodies: Many are reactive with human LGR5, while some cross-react with other species including mouse, rat, and cynomolgus macaque

• Therapeutic antibody formats: Including antibody-drug conjugates (ADCs), bispecific T-cell engagers (BiTEs), and chimeric antigen receptor (CAR) constructs

Over 700 anti-LGR5 antibodies from more than 30 different suppliers are available for various applications such as Western blot, ELISA, Flow Cytometry, IHC, and Immunofluorescence .

How should researchers select the appropriate LGR5 antibody for their specific application?

Selecting the appropriate LGR5 antibody requires consideration of several factors:

• Application compatibility: Ensure the antibody has been validated for your specific application (Western blot, IHC, flow cytometry, etc.)

• Epitope location: For detecting cell surface LGR5, choose antibodies targeting the extracellular domain

• Species reactivity: Verify cross-reactivity with your experimental model organism (human, mouse, rat, etc.)

• Clonality: Monoclonal antibodies offer higher specificity while polyclonals may provide stronger signals in certain applications

• Validation data: Review published literature and supplier validation data showing specificity testing against related proteins like LGR4 and LGR6

For example, when studying LGR5 in live cells, antibodies targeting the N-terminal extracellular domain (like those described in search result ) are preferable as they can bind the native conformation of the protein without requiring cell fixation and permeabilization.

What are the optimal protocols for LGR5 immunohistochemistry in tissue samples?

For effective LGR5 detection in tissue samples by immunohistochemistry, follow this optimized protocol:

Sample preparation:

• Fix tissues in 10% neutral-buffered formalin

• Process and embed in paraffin

• Section at 4-5 μm thickness

Staining protocol:

• Deparaffinize and rehydrate sections

• Perform antigen retrieval with 10 mM citrate buffer (pH 6.0)

• Block endogenous peroxidase with 3% H₂O₂ in PBS for 4 minutes

• Block endogenous biotin using an Avidin/Biotin Blocking Kit

• Block non-specific binding with 10% donkey serum in 3% BSA/PBS

• Incubate with primary anti-LGR5 antibody (4 μg/ml) for 60 minutes at room temperature

• Apply biotinylated secondary antibody (e.g., biotinylated donkey anti-rabbit IgG) for 30 minutes

• Treat with Vectastain ABC Elite HRP for 30 minutes

• Visualize with metal-enhanced DAB for 5 minutes

• Counterstain with Mayer's hematoxylin

Critical considerations:

• Include positive controls (e.g., intestinal crypts) and negative controls (isotype antibody)

• Evaluate slides in a double-blinded fashion using established scoring criteria

• For multi-label studies, carefully select compatible detection systems

This protocol has been validated for detecting LGR5 in various tissue types including colorectal cancer samples .

How can researchers effectively validate LGR5 antibody specificity?

Thorough validation of LGR5 antibodies is critical for reliable results:

Western blot validation:

• Test against cells overexpressing LGR5 (e.g., transfected HEK293T cells)

• Compare with non-transfected controls and cells expressing related proteins (LGR4, LGR6)

• Verify the expected molecular weight (~100 kDa)

• Perform peptide competition assays with specific epitope fragments

Flow cytometry validation:

• Compare staining patterns between LGR5-expressing and non-expressing cells

• Use isotype controls at matching concentrations

• Confirm specificity through blocking peptides (e.g., Frag1A as described in search result )

• Analyze co-expression with established markers in relevant cell populations

Immunofluorescence validation:

• Co-localize with tagged LGR5 constructs (e.g., LGR5-GFP)

• Demonstrate absence of staining in knockout or knockdown models

• Compare subcellular localization patterns with established literature

For example, search result describes validating antibody specificity by testing against human LGR5, cynomolgus LGR5, and related human LGR4, LGR6, and murine Lgr4/Lgr5 expressed in HEK293T cells, demonstrating specific detection of only human and cynomolgus LGR5.

What are the best methods for detecting LGR5 in cell populations by flow cytometry?

For optimal LGR5 detection by flow cytometry:

Sample preparation:

• Harvest cells using gentle dissociation methods (e.g., non-enzymatic cell dissociation solutions)

• For tissues, prepare single-cell suspensions using appropriate tissue-specific dissociation protocols

• Maintain cold conditions throughout to preserve cell surface antigens

Staining protocol:

• Block Fc receptors with appropriate blocking solution

• Stain with fluorophore-conjugated anti-LGR5 or primary/secondary antibody combinations

• Include viability dye to exclude dead cells

• Add appropriate compensation controls

Critical parameters:

• Antibody concentration: Typically 1-10 μg/ml, titrate for optimal signal-to-noise ratio

• Staining time: 30-60 minutes on ice

• Washing steps: Gentle but thorough to reduce background

Essential controls:

• Isotype control matched to primary antibody

• FMO (Fluorescence Minus One) controls

• Positive control (LGR5-transfected cells)

• Negative control (untransfected cells)

For example, search result shows flow cytometry detection of LGR5 in NS0 mouse cell line transfected with human LGR5 and eGFP, comparing specific staining with isotype control and demonstrating clear separation of positive and negative populations.

What are the critical parameters for optimizing Western blot detection of LGR5?

Successfully detecting LGR5 by Western blot requires attention to several critical parameters:

Sample preparation:

• Use appropriate lysis buffers containing detergents (e.g., RIPA buffer with 1% NP-40 or Triton X-100)

• Include protease inhibitors to prevent degradation

• For membrane proteins like LGR5, avoid excessive heating (65°C for 5 minutes instead of boiling)

• Consider membrane fraction enrichment for enhanced detection

Electrophoresis and transfer conditions:

• Use gradient gels (4-12%) for optimal resolution of the 100 kDa LGR5 protein

• Transfer to PVDF membranes (preferred over nitrocellulose for high molecular weight proteins)

• Use low SDS transfer buffer and longer transfer times (90-120 minutes) or semi-dry transfer systems

• Consider wet transfer at 4°C overnight for large proteins

Antibody incubation:

• Recommended dilutions: 1:500-1:1000 for most anti-LGR5 antibodies

• Block with 5% non-fat milk or BSA (depending on antibody recommendations)

• Incubate primary antibody overnight at 4°C for optimal binding

• Use validated secondary antibody at appropriate dilution (typically 1:2000-1:5000)

Signal detection:

• Use enhanced chemiluminescence (ECL) detection with sufficient sensitivity

• Adjust exposure times to prevent oversaturation while capturing authentic signals

• Consider using signal enhancers for low-abundance samples

Troubleshooting weak signals:

• Increase protein loading (up to 50-75 μg per lane)

• Reduce washing stringency

• Increase antibody concentration or incubation time

• Use signal amplification systems

This approach has been validated for detecting the canonical 100 kDa LGR5 protein in various tissue samples and cell lines .

How should researchers approach the isolation and characterization of LGR5-positive cells?

Isolating and characterizing LGR5-positive cells requires a systematic approach:

Cell isolation strategies:

• Flow cytometry-based sorting:

  • Use antibodies against the extracellular domain of LGR5

  • Implement gentle sorting parameters to maintain viability

  • Include viability dyes to exclude dead cells

  • Consider double-sorting for higher purity

• Magnetic-activated cell sorting (MACS):

  • Generally gentler than FACS, potentially preserving stem cell properties

  • May yield higher cell numbers but with lower purity

• Reporter systems (when applicable):

  • LGR5-GFP knock-in systems provide alternative isolation methods

  • Useful for animal models but less applicable to primary human samples

Functional characterization:

• In vitro stem cell assays:

  • Sphere formation efficiency in low-attachment conditions

  • Organoid formation capacity

  • Colony formation efficiency

  • Differentiation potential assessments

• Molecular characterization:

  • Gene expression profiling (qPCR for stem cell markers)

  • Assessment of Wnt pathway activity

  • Analysis of self-renewal and differentiation markers

• In vivo assays:

  • Xenotransplantation at limiting dilutions to assess tumor-initiating capacity

  • Lineage tracing studies (in appropriate models)

Validation of LGR5-positivity:

• Confirm LGR5 expression in sorted populations by alternative methods (qPCR, Western blot)

• Compare with established LGR5+ model systems

• Correlate LGR5 expression with functional stem cell properties

These approaches have been successfully applied to isolate and characterize LGR5+ cancer stem cells from colorectal cancer, hepatocellular carcinoma, and pre-B-ALL samples .

What controls are essential when using LGR5 antibodies for experimental procedures?

Implementing appropriate controls is critical for reliable interpretation of results with LGR5 antibodies:

Positive controls:

• Cell lines with confirmed high LGR5 expression (e.g., NALM6, LoVo cells)

• Tissues known to express LGR5 (e.g., intestinal crypts)

• LGR5-transfected cells (e.g., HEK293T cells overexpressing LGR5)

Negative controls:

• Cell lines lacking LGR5 expression (e.g., untransfected HEK293T cells)

• Tissues with minimal/no LGR5 expression

• Isotype control antibodies at matching concentrations

• Secondary antibody-only controls

Specificity controls:

• Pre-incubation with blocking peptide (e.g., Frag1A as described in search result )

• Non-binding antibody variant (e.g., α-LGR5v6 mentioned in search result )

• siRNA or CRISPR knockout validation where feasible

Application-specific controls:

• For Western blot: Molecular weight markers, loading controls

• For IHC/ICC: Adjacent sections with isotype control

• For flow cytometry: FMO controls, viability dyes

• For functional assays: Non-functional antibody variants (e.g., α-LGR5-ADC NC)

For example, search result describes using a non-binding version of an antibody (α-LGR5v6) generated during the humanization process as a negative control for binding studies, and using a non-cleavable version of an antibody-drug conjugate (α-LGR5-ADC NC) as a specificity control for cell killing assays.

How can LGR5 antibodies be developed into effective cancer therapeutics?

Developing LGR5 antibodies as cancer therapeutics involves several strategic approaches:

Antibody-Drug Conjugates (ADCs):

• Antibody selection:

  • Target the extracellular domain of LGR5 for accessibility

  • Ensure high specificity to avoid off-target effects

  • Select antibodies with optimal internalization kinetics

• Linker-payload optimization:

  • Cleavable linkers have shown superior efficacy compared to non-cleavable versions (α-LGR5-ADC vs. α-LGR5-ADC NC)

  • Drug-to-antibody ratio (DAR) optimization (typically 3.5 for MMAE conjugates)

  • Selection of appropriate cytotoxic payloads

• Efficacy evaluation:

  • In vitro cytotoxicity against LGR5-high vs. LGR5-low cell lines

  • In vivo tumor regression studies in xenograft models

  • Assessment of safety profile and therapeutic window

Research has demonstrated that α-LGR5-ADC effectively kills LGR5+ cancer cells with an EC50 of approximately 10 nM and has shown potent anti-tumor efficacy in a murine model of human NALM6 pre-B-ALL, reducing tumor burden to less than 1% of control treatment .

Bispecific T-cell Engagers (BiTEs):

• Design considerations:

  • Optimal architecture for T-cell recruitment

  • Balanced affinity for LGR5 vs. CD3

  • Size and stability optimization

• Effectiveness:

  • α-LGR5-BiTE treatment has shown moderate efficacy, with approximately twofold reduction in tumor burden in pre-B-ALL models

  • May be further optimized through affinity engineering

Chimeric Antigen Receptor (CAR) T-cells:

• Design elements:

  • LGR5-specific single-chain variable fragments (scFvs)

  • Selection of appropriate costimulatory domains

  • Vector design and T-cell manufacturing considerations

• Performance metrics:

  • α-LGR5-CAR-T cells have demonstrated specific and potent killing of LGR5+ cancer cells in vitro

  • In vivo studies showed a fourfold decrease in pre-B-ALL tumor burden relative to controls

These therapeutic modalities represent promising approaches to targeting LGR5-expressing cancers, with ADCs currently showing the most robust efficacy in preclinical models .

How can researchers distinguish between different pools of LGR5 protein in experimental systems?

Distinguishing between different pools of LGR5 protein requires sophisticated approaches:

Subcellular localization analysis:

• Confocal microscopy with compartment markers:

  • Co-staining with membrane markers (Na+/K+ ATPase, WGA)

  • Golgi markers (GM130, TGN46)

  • Endosomal/lysosomal markers (EEA1, LAMP1)

  • Use z-stack imaging to confirm true co-localization

• Subcellular fractionation:

  • Separate membrane, cytosolic, and organelle fractions

  • Western blot analysis of fractions with LGR5 antibodies

  • Include fraction-specific markers as controls

Functional pools characterization:

• Active vs. inactive forms:

  • Surface biotinylation to quantify membrane-localized fraction

  • Co-immunoprecipitation with pathway components (R-spondins, Frizzled, LRP6)

  • Phosphorylation status assessment of associated signaling molecules

• Dynamic trafficking studies:

  • Pulse-chase experiments with labeled antibodies

  • Photoactivatable or photoconvertible LGR5 fusion proteins

  • Live-cell imaging to track protein movement

Post-translational modification analysis:

• Glycosylation status (PNGase F treatment)

• Phosphorylation patterns (phospho-specific antibodies or mass spectrometry)

• Ubiquitination status (affects degradation and trafficking)

Advanced molecular techniques:

• Proximity ligation assays to detect specific LGR5 interactions

• FRET/BRET approaches to study real-time molecular associations

• Super-resolution microscopy for nanoscale localization patterns

These approaches help distinguish between functionally relevant LGR5 pools (active surface receptors) and processing/storage pools (Golgi, endosomes), providing deeper insights into LGR5 biology in normal and cancer cells.

What approaches can resolve contradictory results when using different LGR5 antibody clones?

When faced with contradictory results using different LGR5 antibody clones, implement these systematic approaches:

Epitope mapping analysis:

• Determine the exact epitopes recognized by each antibody clone

  • Some target N-terminal regions (aa 1-101)

  • Others target mid-protein regions (aa 250-550)

• Assess epitope accessibility in different experimental conditions

• Evaluate potential epitope masking by protein interactions or modifications

Clone-specific validation:

• Test each clone against:

  • Overexpression systems with tagged LGR5

  • Knockout or knockdown models

  • Purified recombinant LGR5 domains

• Binding kinetics analysis:

  • Compare affinity constants (Kd values) for different clones

  • For example, search result shows Kd values ranging from 0.76 to 1.4 nM for different α-LGR5 clones

Orthogonal validation approaches:

• mRNA expression analysis (RT-qPCR, in situ hybridization)

• Mass spectrometry validation of protein identity

• Functional assays (e.g., TOPflash activity assays)

• Combined antibody approaches using antibodies targeting different epitopes

Multi-condition testing:

• Systematically compare antibody performance across:

  • Different fixation methods

  • Various permeabilization protocols

  • Range of antigen retrieval techniques

  • Multiple detection systems

Through comprehensive validation and careful consideration of technical variables, researchers can identify the most reliable antibodies for specific applications and experimental conditions.

How should researchers monitor and validate the internalization dynamics of LGR5 antibodies for ADC development?

Monitoring LGR5 antibody internalization is crucial for ADC development:

Quantitative internalization assays:

• Flow cytometry-based methods:

  • Surface quenching assays (pH-sensitive fluorophores)

  • Acid wash techniques to remove surface-bound antibodies

  • Time-course analysis at multiple timepoints (15, 30, 60, 120 minutes)

• Confocal microscopy approaches:

  • Live-cell imaging with fluorescently-labeled antibodies

  • Co-localization with endosomal/lysosomal markers

  • 3D reconstruction to confirm complete internalization

• Biochemical methods:

  • Surface biotinylation followed by streptavidin pulldown

  • Protease protection assays

  • ELISA-based internalization assays

Critical parameters to evaluate:

• Internalization rate:

  • Half-time to internalization

  • Percentage internalized at steady state

  • Comparison with benchmark internalizing antibodies

• Intracellular trafficking:

  • Endosomal escape efficiency

  • Lysosomal delivery kinetics (critical for cleavable linkers)

  • Recycling vs. degradation fate

• Factors affecting internalization:

  • Antibody affinity influence (research shows Kd values between 0.76-2.7 nM for various α-LGR5 clones)

  • Effect of bivalent vs. monovalent binding

  • Impact of antibody conjugation on trafficking

Correlation with ADC efficacy:

• Compare internalization metrics with cytotoxicity data

• Evaluate linker stability in different cellular compartments

• Assess payload release kinetics in relation to trafficking patterns

Search result demonstrates that α-LGR5 antibodies are rapidly internalized by LGR5-overexpressing cell lines, making them suitable candidates for ADC development. The superior efficacy of cleavable-linker ADCs (α-LGR5-ADC) compared to non-cleavable versions (α-LGR5-ADC NC) highlights the importance of proper internalization and intracellular processing for effective ADC function.

What are the common challenges in detecting LGR5 in experimental systems and how can they be addressed?

Researchers frequently encounter these challenges when detecting LGR5:

Challenge 1: Low expression levels

• Solution: Use signal amplification methods (tyramide signal amplification for IHC, high-sensitivity ECL for Western blot)

• Approach: Employ cell enrichment strategies before analysis

• Technique: Extend antibody incubation times (overnight at 4°C)

Challenge 2: Epitope masking

• Solution: Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

• Approach: Use antibodies targeting different epitopes

• Technique: Optimize fixation protocols (avoid overfixation)

Challenge 3: Nonspecific binding

• Solution: Implement more stringent blocking (5% BSA, 10% serum)

• Approach: Include absorption controls with specific blocking peptides (e.g., Frag1A)

• Technique: Use highly specific monoclonal antibodies with validated specificity

Challenge 4: Distinguishing membrane vs. cytoplasmic staining

• Solution: Use confocal microscopy with membrane markers

• Approach: Implement subcellular fractionation for Western blot analysis

• Technique: Compare surface staining (non-permeabilized) with total staining (permeabilized)

Challenge 5: Cross-reactivity with related proteins

• Solution: Validate antibody specificity against LGR4 and LGR6 expression

• Approach: Confirm findings with genetic approaches (siRNA, CRISPR)

• Technique: Use blocking peptides specific to LGR5 epitopes

Challenge 6: Isoform detection

• Solution: Review antibody epitope location relative to known splice variants

• Approach: Use multiple antibodies targeting different regions

• Technique: Correlate protein detection with mRNA expression of specific variants

Addressing these challenges through rigorous controls and optimized protocols ensures more reliable detection and characterization of LGR5 in research applications.

How can researchers validate that their antibody truly detects LGR5 and not related family members?

Validating LGR5 antibody specificity against related family members requires a multi-faceted approach:

Comparative expression analysis:

• Overexpression systems:

  • Test antibodies against cells overexpressing LGR5, LGR4, and LGR6

  • Use tagged constructs for parallel detection

  • Compare staining/blotting patterns between family members

• Western blot analysis:

  • Run lysates from cells expressing different LGR family members

  • Compare band patterns and molecular weights

  • Perform peptide competition assays

Genetic validation:

• Knockdown/knockout approaches:

  • siRNA or CRISPR-based depletion of LGR5

  • Confirm loss of signal with antibody

  • Test against LGR4/LGR6 knockdowns as controls

• Heterologous expression:

  • Express LGR5 in cells lacking endogenous expression

  • Compare signal between transfected and non-transfected cells

  • Express sequence variants to map epitope requirements

Cross-reactivity testing:

• Species cross-reactivity:

  • Test against human, mouse, rat, and other relevant species

  • Compare sequence conservation at epitope regions

  • Search result shows some antibodies specifically detect human and cynomolgus LGR5 but not murine Lgr5

• Domain-specific detection:

  • Use recombinant protein fragments (e.g., Frag1A and Frag1B)

  • Perform ELISA or SPR binding assays

  • Determine epitope specificity through binding kinetics analysis

• Immunoprecipitation analysis:

  • Pull down with anti-LGR5 antibody

  • Analyze precipitated proteins by mass spectrometry

  • Check for presence of related family members

For example, search result demonstrates that α-LGR5 antibody clones 1-4 detect human and cynomolgus LGR5 but show no immunoreactivity to murine Lgr4 and Lgr5 or the closely related human LGR4 and LGR6 in Western blot analysis of lysates from transfected HEK293T cells.