LGALS3BP Antibody

What is LGALS3BP and what cellular functions does it have?

LGALS3BP is a glycosylated protein with a calculated molecular weight of 65 kDa (585 amino acids), though it typically appears as 65-90 kDa in Western blots due to extensive post-translational modifications . Functionally, LGALS3BP serves as a negative regulator of NF-κB activation and proinflammatory cytokine production . Research demonstrates that it interacts with transforming growth factor β-activated kinase 1 (TAK1), inhibiting its phosphorylation and suppressing its kinase activity . In cancer biology, LGALS3BP is involved in various tumoral progression mechanisms including invasion, migration, immune evasion, and metastatic dissemination .

When designing experiments to study LGALS3BP function, researchers should consider its role both as a cellular protein and as a secreted factor that can act through autocrine and paracrine mechanisms. Immunoprecipitation approaches targeting TAK1 can help confirm the interaction with LGALS3BP in your experimental system.

What applications are LGALS3BP antibodies validated for?

LGALS3BP antibodies have been validated for multiple experimental applications including:

When selecting an antibody for your specific application, consider whether the antibody has been validated in similar sample types. The 10281-1-AP antibody, for example, has been cited in numerous publications spanning diverse applications .

What tissue/cell types show high LGALS3BP expression?

LGALS3BP expression varies across tissues and is particularly elevated in cancer contexts. Expression patterns include:

Normal tissues/cells:

• Human fetal brain tissue

• Human milk

• Human plasma

• Various cell lines including HeLa, COLO 320, HEK-293, HepG2, and A549

Cancer tissues:

• Glioblastoma multiforme (GBM), with significantly higher expression than normal brain tissue

• Adenoid cystic carcinoma (ACC), showing heterogeneous expression patterns

• Melanoma and neuroblastoma, with elevated expression

• Human esophageal, breast, and colon cancer tissues

When studying LGALS3BP expression, it's important to include appropriate positive controls from this list. Immunohistochemical analysis has revealed that expression can be heterogeneous within tumor samples, suggesting the need for comprehensive tissue assessment rather than limited sampling .

How should I optimize antibody dilutions for detecting LGALS3BP?

Optimizing antibody dilutions is critical for achieving specific signal while minimizing background. For LGALS3BP detection, recommended dilution ranges are:

Methodological considerations for optimization:

• Perform a titration series around the recommended range (e.g., 1:500, 1:1000, 1:2000, 1:4000, 1:8000 for WB)

• Include both positive controls (e.g., HepG2 cells) and negative controls

• For IHC, test both suggested antigen retrieval methods: TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0

• Evaluate signal-to-noise ratio at each dilution rather than simply selecting the strongest signal

• For challenging samples, consider extending incubation times rather than increasing antibody concentration to improve signal quality

Remember that optimal dilution is sample-dependent; titration in each experimental system is essential for obtaining reliable results .

What is the molecular weight range for LGALS3BP detection on Western blots?

When performing Western blot analysis of LGALS3BP, researchers should be aware of the following molecular weight considerations:

• Calculated molecular weight: 65 kDa (585 amino acids)

• Observed molecular weight range: 65-90 kDa

• The discrepancy between calculated and observed weight is primarily due to extensive glycosylation

Methodological recommendations:

• Use molecular weight markers that span 50-100 kDa range

• Run gels with sufficient resolution in the 65-90 kDa region (8-10% polyacrylamide gels are suitable)

• Be prepared for potential variation in apparent molecular weight across different sample types due to differential glycosylation patterns

• For cleaner detection, consider longer wash steps after primary and secondary antibody incubations

• If multiple bands appear, enzymatic deglycosylation (e.g., with PNGase F) can help confirm the specific LGALS3BP band

These considerations ensure accurate identification of LGALS3BP in Western blot applications.

How does LGALS3BP expression differ between normal and cancer tissues?

LGALS3BP shows significant expression differences between normal and cancer tissues, making it a protein of interest for cancer diagnostics and therapeutics:

• Cancer overexpression: LGALS3BP is "largely overexpressed in tumor tissue compared to the non-neoplastic counterpart," as demonstrated across multiple cancer types .

• Specific cancer findings:

  • Glioblastoma multiforme (GBM): Immunohistochemical analysis shows high LGALS3BP expression compared to normal brain tissue

  • Adenoid cystic carcinoma (ACC): Heterogeneous expression observed in both PDX models and patient tumor samples

  • Multiple other cancers including melanoma and neuroblastoma show elevated expression

• Methodological approaches for comparative analysis:

  • Paired analysis of tumor and adjacent normal tissue from the same patient

  • Tissue microarray (TMA) analysis across multiple patients

  • Quantitative image analysis of immunohistochemistry staining intensity

  • Correlation of protein expression with patient outcomes and clinical parameters

• Vesicular vs. total protein: In GBM patients, "the amount of vesicular but not total circulating protein is increased" compared to healthy donors , suggesting the importance of analyzing both total and vesicle-associated LGALS3BP.

This differential expression pattern provides both diagnostic opportunities and therapeutic rationale for targeting LGALS3BP in cancer.

What are the best sample preparation methods for LGALS3BP detection in different applications?

Optimal sample preparation is crucial for reliable LGALS3BP detection across different experimental applications:

For Western Blot analysis:

• Lysis buffer: RIPA buffer containing protease inhibitors is suitable for most applications

• For phosphorylation studies (e.g., TAK1 interaction), include phosphatase inhibitors

• Process samples quickly and maintain cold temperatures to prevent protein degradation

• For secreted LGALS3BP, concentrate cell culture supernatants using centrifugal filters or TCA precipitation

For Immunohistochemistry (IHC):

• Fixation: 10% neutral buffered formalin fixation for 24-48 hours

• Antigen retrieval: TE buffer pH 9.0 is recommended as the primary method, with citrate buffer pH 6.0 as an alternative

• Blocking: Use 3-5% normal serum or BSA to reduce background

• For multiplexed staining, carefully select antibodies from different host species

For Immunoprecipitation (IP):

• Gentler lysis buffers (e.g., NP-40 or CHAPS-based) help preserve protein interactions

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

• For co-IP studies targeting TAK1 interactions, consider crosslinking approaches

For Extracellular Vesicle (EV) analysis:

• Isolation methods: Differential ultracentrifugation, size-exclusion chromatography, or precipitation methods

• Validation: Confirm EV isolation using markers like CD63, CD9, and CD81

• Characterization: Nanoparticle tracking analysis and electron microscopy

• Controls: Include detergent controls to confirm vesicular nature of the signal

These optimized methods will enhance detection sensitivity and specificity across applications.

How can I validate the specificity of a LGALS3BP antibody?

Validating antibody specificity is essential for generating reliable data. For LGALS3BP antibodies, a comprehensive validation approach should include:

• Genetic validation approaches:

  • LGALS3BP knockout cells/tissues as negative controls

  • CRISPR-Cas9 generated LGALS3BP-knockout mice have been reported and provide excellent validation models

  • siRNA or shRNA knockdown of LGALS3BP shows dose-dependent reduction in signal

• Biochemical validation methods:

  • Peptide competition assays where pre-incubation with immunizing peptide abolishes specific signal

  • Immunoprecipitation followed by mass spectrometry to confirm identity

  • Deglycosylation experiments to confirm glycoprotein identity based on mobility shift

• Multiple antibody approach:

  • Compare results using antibodies targeting different LGALS3BP epitopes

  • Consistent results with different antibodies increase confidence in specificity

• Expression pattern validation:

  • Confirm detection in known positive control samples (HeLa, HepG2 cells)

  • Verify molecular weight range (65-90 kDa) in Western blots

  • Correlate protein expression with mRNA levels using qPCR

• Application-specific controls:

  • For IHC/IF: Include isotype controls and primary antibody omission controls

  • For WB: Include positive control lysates from cells with confirmed expression

  • For IP: Perform parallel IPs with non-specific IgG

Implementing these validation strategies will establish confidence in antibody specificity and experimental results.

What controls should I use when studying LGALS3BP expression?

Proper experimental controls are essential for accurate interpretation of LGALS3BP expression studies:

Positive Controls:

• Cell lines: HeLa, COLO 320, HEK-293, HepG2, and A549 cells all express LGALS3BP and serve as reliable positive controls

• Tissues: Human fetal brain, milk, and plasma samples have confirmed LGALS3BP expression

• Cancer tissues: Esophageal, breast, and colon cancer tissues show robust expression

Negative Controls:

• LGALS3BP knockout models: CRISPR-Cas9 generated LGALS3BP-knockout mice provide ideal negative controls

• Antibody controls: Isotype-matched IgG or pre-immune serum in place of primary antibody

• Peptide competition: Primary antibody pre-incubated with immunizing peptide

Expression Modulation Controls:

• Overexpression: Transient transfection with LGALS3BP expression vectors

• Knockdown: siRNA or shRNA targeting LGALS3BP with scrambled sequences as controls

• LPS stimulation: For NF-κB pathway studies, compare with and without LPS treatment

Technical Controls:

• Loading controls: β-actin or GAPDH for Western blots

• Microscopy controls: DAPI nuclear counterstain for IHC/IF

• Quantification controls: Standard curves for ELISA-based quantification

These multi-layered controls ensure reliable interpretation of LGALS3BP expression data across experimental contexts.

How does LGALS3BP regulation relate to NF-κB signaling pathways?

LGALS3BP functions as a significant negative regulator of NF-κB signaling through specific molecular mechanisms:

• TAK1 interaction mechanism:

  • LGALS3BP directly interacts with transforming growth factor β-activated kinase 1 (TAK1)

  • This interaction inhibits TAK1 phosphorylation, suppressing its kinase activity

  • Reduced TAK1 activity leads to decreased protein stability and dampened downstream signaling

• Impact on inflammatory signaling:

  • LGALS3BP inhibits NF-κB activation in response to lipopolysaccharide (LPS) stimulation

  • This results in decreased production of proinflammatory cytokines including IL-6, TNF-α, and IL-1β

  • Both mRNA expression and protein secretion of these cytokines are affected

• Experimental evidence:

  • LGALS3BP-deficient mice show enhanced sensitivity to LPS-induced endotoxin shock

  • These mice exhibit shorter survival time and increased levels of proinflammatory cytokines

  • Overexpression of LGALS3BP in both wild-type and knockout MEFs suppresses NF-κB luciferase activity

  • Similar effects were observed in RAW264.7 macrophage cells

• Experimental approaches to study this pathway:

  • NF-κB reporter assays (luciferase) to measure activation

  • Immunoblotting for phosphorylated TAK1 and downstream components

  • Co-immunoprecipitation to confirm LGALS3BP-TAK1 interaction

  • Cytokine ELISAs and qPCR to measure inflammatory outputs

This regulatory role suggests LGALS3BP may have therapeutic potential for inflammatory conditions beyond its applications in cancer.

What are the challenges in targeting LGALS3BP in therapeutic applications?

Developing LGALS3BP-targeted therapeutics presents several complex challenges that researchers must address:

• Expression heterogeneity:

  • LGALS3BP shows heterogeneous expression across patients, as documented in adenoid cystic carcinoma

  • Variability may limit therapeutic efficacy in low-expressing subpopulations

  • Patient stratification based on expression levels may be necessary

• Protein characteristics challenges:

  • As a secreted glycoprotein rather than a membrane-bound receptor, targeting strategies differ from traditional approaches

  • Extensive glycosylation (causing observed 65-90 kDa molecular weight variation) may affect antibody recognition

  • Glycosylation patterns may vary between patients and cancer types

• Physiological function considerations:

  • LGALS3BP's role in negative regulation of NF-κB signaling suggests potential immune-related adverse effects

  • Long-term inhibition might increase inflammatory responses

  • Differential targeting of tumor-associated vs. normal LGALS3BP may be necessary

• Technical and development challenges:

  • For antibody-drug conjugates, controlled conjugation chemistry and optimal drug-to-antibody ratio are critical

  • "Generation of stable cell line producing the engineered 1959-sss antibody as well as scale-up of the site-specific linker-less conjugation process" presents manufacturing challenges

  • For brain tumors, blood-brain barrier penetration must be considered

• Preclinical model limitations:

  • Limited number of PDX models for efficacy testing, as noted in ACC research where "only one PDX model was used for therapeutic studies"

  • Translating preclinical efficacy to clinical outcomes remains uncertain

Despite these challenges, promising results have been obtained with anti-LGALS3BP ADCs, showing "long-lasting response... in 100% of treated animals" in an ACC model and "potent and dose-dependent antitumor activity" in GBM models .

How can LGALS3BP antibodies be used to study extracellular vesicle biology?

LGALS3BP antibodies provide valuable tools for investigating extracellular vesicle (EV) biology, particularly in cancer research:

• Vesicular LGALS3BP as cancer biomarker:

  • "Compared with healthy donors, the amount of vesicular but not total circulating protein is increased" in glioblastoma patients

  • "Plasma vesicular LGALS3BP levels correlate with glioma tumour grade, making this protein a potential biomarker for early detection"

  • Research has shown that "LGALS3BP can be used for liquid biopsy as a marker of disease" in GBM models

• Methodological approaches for EV isolation and analysis:

  • Differential ultracentrifugation: Sequential centrifugation steps (300g, 2000g, 10,000g, 100,000g)

  • Size-exclusion chromatography: Separate EVs based on size using specialized columns

  • Density gradient separation: Distinguish EV subpopulations with different densities

  • Characterization: Nanoparticle tracking analysis, electron microscopy, and Western blotting

• LGALS3BP detection in EVs:

  • Western blotting of EV lysates using anti-LGALS3BP antibodies

  • ELISA-based quantification for high-throughput analysis

  • Immunoelectron microscopy for visualization of LGALS3BP on individual EVs

  • Flow cytometry using bead-based capture systems for EV analysis

• Experimental design considerations:

  • Include EV markers (CD9, CD63, CD81) to confirm vesicle isolation

  • Use negative markers (GM130, calnexin) to exclude cellular contamination

  • Compare multiple isolation methods to ensure consistent results

  • Match isolation protocols between patient samples and controls

This approach allows researchers to leverage LGALS3BP as a biomarker for minimally invasive liquid biopsy applications, potentially improving early cancer detection and monitoring.

What experimental approaches can assess LGALS3BP's role in immune evasion?

Investigating LGALS3BP's potential role in cancer immune evasion requires specialized experimental approaches:

• Tumor-immune cell interaction studies:

  • Co-culture systems: Compare wild-type vs. LGALS3BP-knockout tumor cells with immune cells

  • Functional assays: Measure T cell proliferation, cytotoxicity, and cytokine production

  • Migration assays: Assess immune cell recruitment in response to LGALS3BP-containing conditioned media

• NF-κB pathway analysis in immune contexts:

  • Given LGALS3BP's role as a negative regulator of NF-κB activation , examine effects on immune cell activation

  • Measure TAK1 phosphorylation in immune cells exposed to recombinant LGALS3BP

  • Compare cytokine production in immune cells from wild-type vs. LGALS3BP-knockout mice

• In vivo immune profiling:

  • Utilize LGALS3BP-knockout mice to assess immune responses to tumor challenge

  • Perform immunophenotyping of tumor-infiltrating lymphocytes in presence/absence of LGALS3BP

  • Evaluate combination approaches with immune checkpoint inhibitors

• Vesicular LGALS3BP immunomodulation:

  • Isolate LGALS3BP-containing EVs from tumor cells

  • Assess effects on dendritic cell maturation and T cell activation

  • Compare immunomodulatory properties of EVs from LGALS3BP-knockout vs. wild-type cells

• Molecular and cellular analysis techniques:

  • Multiplex cytokine analysis focused on IL-6, TNF-α, and IL-1β

  • Flow cytometry to assess immune cell activation markers

  • Single-cell RNA sequencing to identify transcriptional changes in immune populations

  • Spatial transcriptomics to map LGALS3BP expression relative to immune infiltration

These approaches can help elucidate whether LGALS3BP contributes to tumor immune evasion, potentially opening new avenues for immunotherapy combinations.

How do post-translational modifications affect LGALS3BP antibody recognition?

Post-translational modifications (PTMs), particularly glycosylation, significantly impact LGALS3BP antibody recognition and must be carefully considered:

• Molecular weight variability due to glycosylation:

  • LGALS3BP has a calculated molecular weight of 65 kDa but is observed at 65-90 kDa in Western blots

  • This substantial variation (~25 kDa) indicates extensive glycosylation

  • Different cell types and tissues may show different apparent molecular weights

• Experimental approaches to address PTM variability:

  • Enzymatic deglycosylation: Treat samples with PNGase F to remove N-linked glycans

  • Compare native vs. deglycosylated forms on Western blots

  • Use multiple antibodies targeting different epitopes to ensure comprehensive detection

  • Include positive control samples with known glycosylation patterns

• Technical considerations for antibody selection:

  • Antibodies targeting glycosylated epitopes may show variable recognition

  • The 10281-1-AP antibody was generated against a LGALS3BP fusion protein (Ag0294) , which may affect epitope presentation

  • For consistent detection, select antibodies targeting protein regions less affected by glycosylation

• Methodological recommendations for consistent detection:

  • Run gradient gels to improve resolution of different glycoforms

  • Consider lectin blotting in parallel to assess glycosylation patterns

  • For quantitative analysis, compare results from antibodies targeting different regions

  • Document the specific molecular weight observed in your experimental system

• Therapeutic implications:

  • For antibody-drug conjugates like 1959-sss/DM4, PTM variability could affect targeting efficiency

  • Selection of antibodies for therapeutic use should prioritize epitopes consistently presented despite PTM variations

Understanding these PTM effects is crucial for reliable detection and effective therapeutic targeting of LGALS3BP.

What are the best methodologies for developing LGALS3BP-targeted antibody-drug conjugates?

Development of effective LGALS3BP-targeted antibody-drug conjugates (ADCs) requires sophisticated methodological approaches, as exemplified by the 1959-sss/DM4 ADC described in the literature:

• Antibody engineering considerations:

  • The 1959-sss antibody is described as an "engineered humanized monoclonal antibody"

  • Humanization reduces immunogenicity for clinical applications

  • Site-specific engineering enables controlled conjugation chemistry

• Target validation methodologies:

  • Comprehensive immunohistochemical analysis across normal and cancer tissues

  • Quantitative assessment of expression levels in target cancers

  • Heterogeneity analysis within tumor samples to predict response rates

  • Confirmation that LGALS3BP is "largely overexpressed in the tumor tissue compared to the non-neoplastic counterpart"

• Strategic payload selection:

  • The 1959-sss/DM4 ADC utilizes SH-DM4, "a highly lipophilic analogue of maytansine"

  • Lipophilic properties enable diffusion into cells and potential bystander effect

  • This is particularly important for LGALS3BP as a secreted rather than membrane-bound target

• Conjugation technology optimization:

  • "Site-specific linker-less conjugation process" provides more homogeneous ADC products

  • Optimizing drug-to-antibody ratio for maximum efficacy while maintaining stability

  • Analytical characterization of ADC homogeneity and stability

• Preclinical evaluation approaches:

  • In vivo imaging using "[89Zr]Zr-DFO-1959" radioimmunoconjugate to confirm tumor targeting

  • Patient-derived xenograft (PDX) models that maintain tumor heterogeneity

  • Efficacy studies demonstrating "durable tumor growth inhibition (TGI) in 100% of animals without observed toxicity"

  • Pharmacokinetic and biodistribution analysis

The 1959-sss/DM4 ADC has demonstrated impressive preclinical results across multiple cancer types including "potent and durable antitumor activity in melanoma, neuroblastoma and glioblastoma" and promising activity in adenoid cystic carcinoma , warranting further clinical development.