LGALS4 Antibody, Biotin conjugated

What is LGALS4 and what are its primary functions in normal and pathological conditions?

LGALS4 (Lectin, Galactoside-binding, Soluble, 4), commonly known as Galectin-4 (Gal-4), is a tandem-repeat galectin consisting of two carbohydrate recognition domains connected by a flexible linker . It's primarily expressed in epithelial cells throughout the gastrointestinal tract, including the antrum, ileum, colon, and rectum .

In normal physiology, Galectin-4:

• Participates in lipid raft stabilization

• Contributes to protein apical trafficking

• Plays roles in wound healing processes

In pathological conditions, particularly cancer:

• Acts as a cancer cell-produced protein abundant in pancreatic ductal adenocarcinoma (PDAC)

• Blocks antitumor immunity by inducing apoptosis in T cells through binding to N-glycosylation residues on CD3ε/δ

• Shows dual roles in different cancers, as it can inhibit colorectal cancer (CRC) cell growth while promoting immune evasion in pancreatic cancer

Understanding these functions helps researchers select appropriate experimental designs when using LGALS4 antibodies for investigating both physiological and pathological processes.

What are the optimal applications for biotin-conjugated LGALS4 antibodies compared to unconjugated versions?

Biotin-conjugated LGALS4 antibodies offer distinct advantages in specific applications:

Biotin-conjugated antibodies are particularly advantageous for:

• Amplification systems using streptavidin-based detection methods, providing enhanced sensitivity for low-abundance Galectin-4 detection

• Multiple labeling experiments where several antibodies must be used simultaneously

• Flow cytometry applications detecting surface-bound Galectin-4, as seen in studies comparing Gal-4 expression between different colorectal cancer cell lines

• Immunohistochemical applications requiring signal amplification, especially in pancreatic cancer tissues where ECM deposits are abundant

For optimal results, researchers should validate the specificity of the biotin-conjugated antibody using known positive controls such as human colon tissue or HT-29 cells .

How should experimental protocols be optimized when using biotin-conjugated LGALS4 antibodies for IHC?

When optimizing immunohistochemistry protocols with biotin-conjugated LGALS4 antibodies, researchers should consider:

Antigen Retrieval:

• For formalin-fixed, paraffin-embedded tissues, use TE buffer pH 9.0 for optimal retrieval

• Alternative: citrate buffer pH 6.0 may be effective for some tissue types

Blocking Steps (Critical for Biotin-Conjugated Antibodies):

• Block endogenous peroxidases using dual enzyme block (e.g., Dako)

• Block non-specific binding with 2.5% normal horse serum (Vector laboratories)

• Critically, block endogenous biotin using a biotin blocking kit:

  • Incubate with streptavidin solution (20 minutes)

  • Wash with TBS (2×1 minute)

  • Incubate with biotin solution (20 minutes)

Antibody Dilution and Incubation:

• Optimal concentration: 5-20 μg/ml in TBS with 5% Superblock and 5% goat serum

• Incubation time: 40-60 minutes at room temperature

Detection System:

• Use HRP-conjugated polymers (30 minutes incubation)

• Optimize DAB substrate incubation for each primary antibody

• Use hematoxylin as a nuclear counterstain

Validation Controls:

• Positive controls: human colon sections (known to express high levels of Galectin-4)

• Negative controls: antibody diluent without primary antibody

• Tissue panel controls: include lymph node sections for comparative analysis

This systematic approach ensures consistent and specific staining patterns when using biotin-conjugated LGALS4 antibodies for IHC applications.

What are the recommended storage and handling conditions for maintaining biotin-conjugated LGALS4 antibody activity?

Proper storage and handling are crucial for maintaining the activity and specificity of biotin-conjugated LGALS4 antibodies:

Storage Temperature:

• Long-term storage: -20°C (stable for one year after shipment)

• Frequent use: 4°C (for up to one month)

• Never freeze RPE-conjugated formats

Buffer Formulation:

Most manufacturers provide these antibodies in a stabilizing buffer containing:

• 0.01M PBS, pH 7.4

• 50% glycerol (cryoprotectant)

• 0.05% Proclin-300 or 0.02-0.03% sodium azide (preservative)

Aliquoting Recommendations:

• Aliquot upon receipt to minimize freeze-thaw cycles

• Use small volume aliquots (20-50 μl) based on typical experiment needs

• Some formats (20 μl sizes) may contain 0.1% BSA for added stability

Avoid Destructive Conditions:

• Minimize freeze-thaw cycles (each cycle can reduce activity by 10-20%)

• Protect from prolonged exposure to light (especially critical for dual-labeled antibodies)

• Keep at recommended temperature during shipping and handling

Stability Assessment:

The thermal stability can be described by loss rate, with high-quality antibodies showing less than 5% loss within the expiration date under appropriate storage conditions .

Following these guidelines will help preserve the functional integrity of biotin-conjugated LGALS4 antibodies for research applications.

How can researchers validate the specificity of biotin-conjugated LGALS4 antibodies?

Validating antibody specificity is essential for reliable experimental results. For biotin-conjugated LGALS4 antibodies, implement these validation strategies:

Positive Control Tissues and Cell Lines:

• Human colon tissue (high endogenous expression)

• Colorectal cell lines with confirmed expression:

  • HT-29 (moderate expression)

  • T84 (high expression)

  • COLO 205 (high expression)

Negative Controls:

• Use IgG from same species at equivalent concentration

• Include tissues known to lack Galectin-4 expression

• Consider HCT-116 cells as negative/low-expressing control

Western Blot Verification:

Perform Western blot analysis to confirm expected molecular weight (36 kDa) in positive control lysates. Western blot under reducing conditions using appropriate immunoblot buffer groups can verify antibody specificity .

Cross-Reactivity Testing:

Test for cross-reactivity with other galectin family members, particularly Galectin-3, -7, and -8, which have structural similarities. High-quality antibodies show no cross-reactivity with recombinant mouse Galectin-1, -3, -4, or -7 .

Antibody Blocking/Competition:

• Pre-incubate antibody with recombinant LGALS4 protein

• Compare staining patterns with and without blocking

• Significant reduction in signal confirms specificity

Knockout/Knockdown Validation:

When possible, validate using LGALS4 knockout or knockdown models, which should show significantly reduced or absent staining compared to wild-type samples .

These comprehensive validation steps ensure that experimental results using biotin-conjugated LGALS4 antibodies are reliable and reproducible.

What methodological approaches can detect surface-bound versus intracellular Galectin-4 using biotin-conjugated antibodies?

Distinguishing between surface-bound and intracellular Galectin-4 requires specific methodological approaches:

Flow Cytometry for Surface-Bound Detection:

• Harvest cells using enzyme-free dissociation buffer to preserve surface proteins

• Wash cells in cold PBS containing 1% BSA

• Incubate with biotin-conjugated anti-LGALS4 antibody (dilution 1:200) for 30 minutes on ice

• Wash cells 3× with cold PBS/BSA

• Incubate with streptavidin-fluorophore conjugate

• Analyze by flow cytometry without permeabilization

This approach has successfully identified surface-bound Galectin-4 in LS-180 and HT-29 cells but not in HCT-116 cells, confirming differential surface expression patterns .

Immunofluorescence for Spatial Localization:

• For surface-bound detection:

  • Culture cells on microscope coverslips

  • Fix with 4% paraformaldehyde (10 minutes, room temperature)

  • Block with 5% goat serum in PBS (no detergent)

  • Incubate with biotin-conjugated LGALS4 antibody

  • Detect with streptavidin-Alexa Fluor 488

• For total Galectin-4 detection:

  • Include permeabilization step (0.1% Triton X-100, 10 minutes)

  • Compare patterns between permeabilized and non-permeabilized samples

Comparative Analysis:

Research has shown that surface-bound Galectin-4 exhibits distinct fluorescence profiles in flow cytometry. Cells with surface-bound Gal-4 (LS-180 and HT-29) form a distinctly different fluorescence peak with increased intensity compared to cells lacking surface expression (HCT-116) .

This differential detection approach provides crucial insights into Galectin-4's localization and function in various cellular contexts, particularly important when studying its role in tumor microenvironments.

How can researchers design experiments to investigate LGALS4's role in immune evasion using biotin-conjugated antibodies?

Designing experiments to investigate LGALS4's role in immune evasion requires a multi-faceted approach:

T-Cell Apoptosis Assay:

• Isolate T cells from peripheral blood or mouse spleen

• Co-culture with cancer cells expressing different levels of LGALS4

• Use biotin-conjugated anti-LGALS4 antibody to:

  • Block LGALS4-CD3 interaction

  • Detect surface-bound LGALS4 by flow cytometry

• Assess T-cell apoptosis using Annexin V/PI staining

• Measure changes in T-cell activation markers (CD69, CD25)

This approach can reveal how LGALS4 binding to N-glycosylation residues on CD3ε/δ induces T-cell apoptosis .

In Vivo Tumor Growth Models:

• Establish orthotopic pancreatic cancer models in immunocompetent and immunodeficient mice

• Treat with biotin-conjugated anti-LGALS4 antibody or control antibody

• Analyze:

  • Tumor growth rate

  • T-cell infiltration (using multicolor immunohistochemistry)

  • Myeloid cell populations

  • Cancer-associated fibroblast subtypes

Research has shown increased T-cell infiltration and prolonged survival in mice with reduced LGALS4 expression, but only in immunocompetent animals, confirming the immune-mediated effect .

Single-Cell RNA Sequencing Analysis:

Combine antibody treatments with scRNA-seq to examine changes in:

• Myeloid compartment composition

• T-cell exhaustion signatures

• Cancer-associated fibroblast subtypes

Studies have shown that reduced LGALS4 expression associates with higher proportions of myofibroblastic CAFs, reduced inflammatory CAFs, and increases in M1 macrophages, T cells, and antigen-presenting dendritic cells .

These experimental approaches provide comprehensive insights into how LGALS4 contributes to immune evasion in cancer microenvironments.

What are the considerations for multiplexed immunofluorescence when using biotin-conjugated LGALS4 antibodies?

Multiplexed immunofluorescence using biotin-conjugated LGALS4 antibodies requires careful planning:

Antibody Panel Design:

When designing multiplexed panels including biotin-conjugated LGALS4 antibodies, consider:

• Species compatibility (avoid antibodies raised in same host)

• Fluorophore selection (minimal spectral overlap)

• Sequential staining for biotin-based detection systems

Sample Preparation Optimization:

• Antigen retrieval conditions:

  • Use TE buffer pH 9.0 for LGALS4 detection

  • Validate compatibility with other target antigens

  • Consider sequential antigen retrieval if necessary

• Blocking strategy:

  • Complete biotin/avidin blocking is essential (20 minutes each component)

  • Follow with protein block containing 10% Superblock TBS and 5% goat serum

  • Include 0.01% Tween-20 to reduce non-specific binding

Sequence Considerations:

When including biotin-conjugated LGALS4 antibody in a multiplex panel:

• Apply the biotin-conjugated LGALS4 antibody first

• Block with streptavidin

• Apply subsequent antibodies with direct fluorescent conjugates

• Use tyramide signal amplification for weaker signals

Validation Protocols:

For each multiplex panel, verify:

• Single-plex controls for each antibody

• FMO (fluorescence minus one) controls

• Absorption controls to confirm specificity

Demonstrated Multiplex Applications:

In pancreatic cancer research, biotin-conjugated LGALS4 antibodies have been successfully used in multiplex panels examining:

• T-cell subtypes (CD4+, CD8+, FOXP3+) in relation to LGALS4 expression

• Myeloid cell infiltration patterns

• Changes in cancer-associated fibroblast populations

This approach allows researchers to simultaneously visualize LGALS4 expression and immune cell populations within the tumor microenvironment.

How should researchers analyze contradictory data regarding LGALS4's role in different cancer types?

Research has revealed seemingly contradictory roles for LGALS4 in different cancer types, requiring careful analytical approaches:

Context-Dependent Effects Analysis:

When analyzing contradictory data, consider that LGALS4 exhibits context-dependent effects:

• In pancreatic cancer: Promotes immune evasion by inducing T-cell apoptosis

• In colorectal cancer: Acts as a tumor suppressor by inhibiting cell proliferation, inducing cell cycle arrest, and promoting apoptosis

Experimental Design for Resolving Contradictions:

• Cell-type specific analysis:

  • Compare LGALS4 expression levels across tissue types

  • Document subcellular localization differences (nuclear, cytoplasmic, surface-bound)

  • Examine glycosylation patterns in different tissues

• Functional domain analysis:

  • Investigate the roles of different carbohydrate recognition domains

  • Design experiments examining the flexible linker region

  • Test truncated protein variants to identify functional elements

Quantitative Expression Analysis:

Research has shown differential LGALS4 expression patterns:

• Underexpressed in LoVo and HCT-116 colorectal cancer cells compared to normal cells

• Abundant in pancreatic tumor extracellular matrix

• Detectable at high levels in patient circulation with PDAC

These differences may explain contradictory functional effects.

Receptor Interaction Studies:

When analyzing contradictory findings, examine LGALS4's interaction with different binding partners:

• CD3ε/δ on T cells (immune suppression)

• Cell cycle regulators like Cyclin B1, CDK1, and Cyclin A2 (anti-proliferative)

• Apoptosis mediators including CASP3, BAX, and CASP9 (pro-apoptotic)

This comprehensive approach helps reconcile apparent contradictions in LGALS4's biological roles across cancer types.

What are the methodological considerations for using biotin-conjugated LGALS4 antibodies in studying cancer-immune interactions?

Studying cancer-immune interactions using biotin-conjugated LGALS4 antibodies requires specific methodological considerations:

Tissue Processing and Preservation:

• Optimal fixation methods:

  • Short fixation times (4-8 hours) in 10% neutral buffered formalin

  • Avoid over-fixation which can mask epitopes

  • Consider fresh-frozen sections for sensitive applications

• Sample collection timing:

  • Account for circadian variations in LGALS4 expression

  • Document treatment history of tissue samples

  • Note inflammatory conditions that may alter expression

Dual Labeling Strategies:

For simultaneous detection of LGALS4 and immune cell markers:

• Apply biotin-conjugated LGALS4 antibody (5-20 μg/ml)

• Detect with streptavidin-HRP and DAB substrate

• Apply heat-mediated antigen retrieval to remove primary antibodies

• Apply immune cell marker antibodies (CD4, CD8, FOXP3)

• Detect with alkaline phosphatase-based visualization system

Flow Cytometry Protocol for Immune Cell Analysis:

When examining LGALS4's effects on immune cells:

• Isolate tumor-infiltrating lymphocytes

• Stain with biotin-conjugated LGALS4 antibody

• Detect with streptavidin-fluorophore

• Co-stain with immune subset markers

• Analyze cell populations by multiparameter flow cytometry

This approach has successfully identified T-cell populations affected by LGALS4 in tumor microenvironments .

Functional Assay Development:

To study LGALS4's immunomodulatory effects:

• Design co-culture systems with defined LGALS4 expression levels

• Use biotin-conjugated antibodies to neutralize or detect LGALS4

• Measure functional outcomes:

  • T-cell activation (IL-2 production, CD69 expression)

  • Cytotoxic activity (target cell killing assays)

  • Cytokine profile changes (multiplex assays)