LOXL2 Antibody, FITC conjugated

What is LOXL2 and why is it a significant research target?

LOXL2 (lysyl oxidase-like 2), also known as WS9-14, belongs to the amine oxidase family whose members play crucial roles in crosslink formation in stromal collagens and elastin. LOXL2 is implicated in cell motility, epithelial-mesenchymal transition (EMT), and tumor development and progression . It functions as a modulator of Snail, providing an additional control mechanism of EMT, making it a significant research target in cancer biology and fibrosis-related conditions . The protein has a calculated molecular weight of 87 kDa, though it is often observed at approximately 100-105 kDa in experimental conditions .

What are the main applications for LOXL2 antibodies in research?

LOXL2 antibodies are utilized across multiple experimental platforms:

The dilution ranges should be experimentally determined for optimal results in each specific testing system .

What is the significance of FITC conjugation for LOXL2 antibodies?

FITC (Fluorescein Isothiocyanate) conjugation provides direct fluorescent labeling of the LOXL2 antibody, eliminating the need for secondary antibody detection in certain applications. The FITC fluorophore has an excitation wavelength of 495 nm and emission at 519 nm . This conjugation is particularly valuable for:

• Flow cytometry applications where direct detection reduces background and simplifies protocols

• Immunofluorescence assays requiring multiplex detection with other antibodies

• Functional assays where direct visualization of antibody binding is beneficial

• Experiments where cross-reactivity with secondary antibodies is problematic

FITC-conjugated antibodies allow for rapid detection and visualization of LOXL2 in experimental systems while maintaining the specificity of the parent antibody .

How should researchers optimize FITC-conjugated LOXL2 antibody use in flow cytometry?

When designing flow cytometry experiments with FITC-conjugated LOXL2 antibodies, researchers should consider:

• Titration: Perform antibody titration to determine optimal concentration, starting with manufacturer's recommendations (typically 1-10 μg/mL) and testing 2-3 dilutions above and below .

• Controls:

  • Include an isotype control (FITC-conjugated human IgG for NBP3-28051F)

  • Use positive controls from validated cell lines expressing LOXL2 (e.g., HepG2, A549, U-87 MG)

  • Include negative controls of cells with low or no LOXL2 expression

• Sample preparation:

  • Use appropriate fixation methods that preserve epitope recognition

  • Consider membrane permeabilization if targeting intracellular LOXL2

  • Block non-specific binding with appropriate buffers

• FITC considerations:

  • Account for potential spectral overlap with other fluorophores

  • Protect from light to prevent photobleaching

  • Consider autofluorescence of samples in the FITC channel

• Data analysis:

  • Set proper compensation if using multiple fluorophores

  • Use median fluorescence intensity rather than mean for more robust quantification

What are the recommended protocols for immunofluorescence using FITC-conjugated LOXL2 antibodies?

For optimal immunofluorescence results with FITC-conjugated LOXL2 antibodies:

• Cell preparation and fixation:

  • Culture cells on appropriate substrates (coverslips, chamber slides)

  • Fix with 4% paraformaldehyde for 15-20 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 if detecting intracellular LOXL2

• Blocking and antibody incubation:

  • Block with 5-10% normal serum in PBS for 30-60 minutes

  • Dilute FITC-conjugated LOXL2 antibody to 1:50-1:500 in blocking buffer

  • Incubate overnight at 4°C or 1-2 hours at room temperature in a humidified chamber

  • Protect from light during all steps involving the FITC-conjugated antibody

• Validated positive controls:

  • HepG2 cells have been validated for LOXL2 detection by IF/ICC

  • Consider U-87 MG human glioblastoma cells which also express LOXL2

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI

  • Mount with anti-fade mounting medium

  • Seal edges of coverslip to prevent drying

• Imaging considerations:

  • Use appropriate filter sets for FITC (excitation 495 nm, emission 519 nm)

  • Adjust exposure settings to prevent photobleaching

  • Capture multiple fields to ensure representative imaging

Validation with appropriate controls is essential, as is comparing results with published literature showing LOXL2 subcellular localization patterns .

How can researchers validate LOXL2 antibody specificity for their experimental system?

Rigorous validation of LOXL2 antibody specificity is critical for experimental integrity:

• Western blot validation:

  • Test the antibody on positive control lysates (A549, A431, HeLa, MDA-MB-453s, HEC-1-B, or U-87 MG cells)

  • Confirm detection at the expected molecular weight (~100-105 kDa)

  • Include negative controls and/or knockdown samples if available

• Knockdown/knockout validation:

  • Compare antibody signal between control and LOXL2 knockdown/knockout samples

  • shRNA-mediated knockdown of LOXL2 has been documented to validate antibody specificity

  • Examine both mRNA expression (qPCR) and protein levels (Western blot) to confirm knockdown efficiency

• Peptide competition assay:

  • Pre-incubate antibody with the immunizing peptide or recombinant LOXL2 protein

  • Compare staining with blocked versus unblocked antibody

  • Signal should be significantly reduced with peptide competition

• Multiple antibody comparison:

  • Test multiple antibodies targeting different LOXL2 epitopes

  • Consistent results across different antibodies increase confidence in specificity

• Functional validation:

  • Assess lysyl oxidase activity in samples with and without antibody treatment

  • Combine with knockdown studies to correlate protein levels with enzymatic activity

The validation steps should be documented in preliminary experiments and included in publications to demonstrate antibody reliability .

What are the current approaches for using LOXL2 antibodies in cancer research?

LOXL2 antibodies are deployed in cancer research through several sophisticated approaches:

• Tumor microenvironment studies:

  • Examination of LOXL2 in extracellular matrix remodeling

  • Analysis of collagen crosslinking in tumor stroma using LOXL2 antibodies to track enzyme localization

  • Investigation of LOXL2's role in creating favorable niches for metastatic cells

• Metastasis investigations:

  • Detection of LOXL2 in circulating tumor cells

  • Evaluation of LOXL2 expression in primary tumors versus metastatic lesions

  • Correlation of LOXL2 levels with invasive potential and metastatic burden

• Therapeutic development:

  • Use of humanized anti-LOXL2 antibodies (similar to simtuzumab) in preclinical models

  • Evaluation of FITC-conjugated antibodies for biodistribution studies

  • Assessment of antibody-drug conjugates targeting LOXL2-expressing cells

• Prognostic marker validation:

  • Immunohistochemical analysis of patient samples using standardized protocols (1:50-1:500 dilution)

  • Correlation of LOXL2 expression with patient outcomes

  • Multiparameter analysis combining LOXL2 with other biomarkers

• EMT and cancer stemness:

  • Investigation of LOXL2's interaction with Snail and its effect on EMT

  • Analysis of LOXL2 in cancer stem cell populations using flow cytometry

  • Tracking LOXL2 dynamics during therapy resistance development

Researchers have observed increased LOXL2 expression in various cancer types, particularly in colon and esophageal cancer, making LOXL2 antibodies valuable tools for cancer biology investigations .

How can researchers effectively use LOXL2 antibodies to study collagen crosslinking and ECM remodeling?

LOXL2 plays a critical role in extracellular matrix (ECM) modification through collagen crosslinking, which can be studied using specialized approaches:

• In vitro collagen gel contraction assays:

  • Preparation of collagen gels with isolated exosomes or recombinant LOXL2

  • Treatment with LOXL2 antibodies to inhibit crosslinking activity

  • Quantification of gel contraction as a measure of crosslinking

  • Comparison between control and LOXL2 knockdown conditions

• Lysyl oxidase activity assays:

  • Measurement of enzymatic activity using fluorometric assays

  • Assessment of LOXL2-specific activity through antibody inhibition studies

  • Correlation of activity with protein levels detected by Western blot

  • Examination of activity under different conditions (e.g., normoxia vs. hypoxia)

• Live cell imaging of ECM remodeling:

  • Use of FITC-conjugated LOXL2 antibodies to track enzyme localization

  • Time-lapse microscopy to monitor dynamic interactions with matrix components

  • Co-localization studies with other ECM proteins and modifiers

• Secretome analysis:

  • Examination of LOXL2 in exosomes using Western blot (observed at ~100-105 kDa)

  • Study of LOXL2 secretion patterns under various stimuli

  • Isolation and characterization of LOXL2-containing extracellular vesicles

• Tissue stiffness correlation:

  • Immunohistochemical analysis of LOXL2 distribution in tissues

  • Correlation with mechanical properties measured by atomic force microscopy

  • Association with collagen organization assessed by second harmonic generation microscopy

Research has shown that LOXL2 knockdown significantly reduces collagen crosslinking activity in exosomes, demonstrating the enzyme's direct role in ECM modification and potential as a therapeutic target in fibrosis-related conditions .

What approaches help resolve contradictory data when working with LOXL2 antibodies?

When faced with inconsistent or contradictory results when using LOXL2 antibodies, researchers should systematically address potential sources of variation:

• Antibody characterization issues:

  • Compare results between different antibody clones (monoclonal vs. polyclonal)

  • Examine epitope locations for potential masking or modification in different contexts

  • Verify antibody performance in multiple applications (WB, IHC, IF) to establish reliability

• Isoform and post-translational modification considerations:

  • LOXL2 has been observed at different molecular weights (87 kDa calculated, 100-105 kDa observed)

  • Investigate potential glycosylation or other post-translational modifications

  • Consider alternative splicing variants that might affect antibody recognition

• Experimental condition standardization:

  • Ensure consistent sample preparation (lysis buffers, fixation methods)

  • Standardize antigen retrieval methods for IHC (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Control cell culture conditions that might affect LOXL2 expression (e.g., hypoxia)

• Multi-method confirmation:

  • Validate protein expression with orthogonal techniques (e.g., mass spectrometry)

  • Combine protein detection with mRNA quantification

  • Use genetic manipulation (siRNA, CRISPR) to confirm signal specificity

• Context-specific expression:

  • Document cell type and tissue-specific variations in LOXL2 expression

  • Consider microenvironmental factors that might regulate LOXL2

  • Examine LOXL2 in relationship with binding partners or functional complexes

When publishing contradictory findings, researchers should thoroughly document methodological details, include all controls, and directly address differences from previous literature with potential explanations for discrepancies .

How can researchers optimize signal-to-noise ratio when using FITC-conjugated LOXL2 antibodies?

Achieving optimal signal-to-noise ratio is critical for clear results with FITC-conjugated LOXL2 antibodies:

• Antibody titration and optimization:

  • Test multiple dilutions to determine optimal concentration

  • For IF/ICC applications, start with 1:50-1:500 dilution range

  • For flow cytometry, perform systematic titration with 2-fold serial dilutions

• Background reduction strategies:

  • Use freshly prepared 4% paraformaldehyde for fixation

  • Implement thorough washing steps (3-5x with PBS-Tween)

  • Include proper blocking (5-10% serum from species unrelated to antibody host)

  • Consider addition of 0.1-0.3% Triton X-100 to reduce non-specific binding

  • Use filtered buffers to remove particulates that may cause artifacts

• Sample-specific considerations:

  • For tissues with high autofluorescence, consider:

    • Treatment with sodium borohydride to reduce aldehyde-induced fluorescence

    • Brief incubation with Sudan Black B (0.1-0.3%) to quench lipofuscin

    • Use of longer wavelength fluorophores if autofluorescence persists

• FITC-specific optimization:

  • Protect from light during all steps to prevent photobleaching

  • Store in dark at 4°C as recommended by manufacturer

  • Consider photobleaching controls in quantitative studies

  • Adjust pH of buffers to optimize FITC fluorescence (pH 8.0-9.0 is optimal)

• Imaging parameters:

  • Optimize exposure time and gain settings

  • Use appropriate filter sets (excitation 495 nm, emission 519 nm)

  • Consider confocal microscopy for improved signal-to-noise ratio

The optimal approach may vary depending on specific sample types, with human tissue samples often requiring more rigorous background reduction techniques compared to cell lines .

What are the critical storage and handling requirements for maintaining FITC-conjugated LOXL2 antibody performance?

FITC-conjugated antibodies require special handling to maintain integrity and performance:

• Temperature considerations:

  • Store at 4°C in the dark as recommended for FITC-conjugated LOXL2 antibodies

  • Non-conjugated LOXL2 antibodies are typically stored at -20°C

  • Avoid repeated freeze-thaw cycles which can degrade both antibody and fluorophore

• Light protection:

  • Store and handle in amber tubes or wrap in aluminum foil

  • Minimize exposure to light during all experimental procedures

  • Work under reduced ambient lighting when possible

  • Consider using red-filtered laboratory lighting for extended procedures

• Buffer compatibility:

  • Maintain appropriate pH (7.3-7.4) for antibody stability

  • PBS with preservatives is typically used for storage

  • Avoid buffers containing primary amines that may react with free FITC

• Preservatives and additives:

  • Standard LOXL2 antibodies often contain 0.02% sodium azide and 50% glycerol

  • FITC-conjugated antibodies may contain 0.05% sodium azide

  • Be aware that sodium azide can inhibit HRP in applications combining with peroxidase detection

• Stability considerations:

  • Non-conjugated antibodies are typically stable for one year after shipment when properly stored

  • FITC-conjugated antibodies may have shorter shelf life due to potential fluorophore degradation

  • Consider aliquoting to minimize freeze-thaw cycles if storing frozen

  • Document lot numbers and reception dates to track potential performance changes

Following these guidelines will help maintain the specificity, sensitivity, and fluorescence intensity of FITC-conjugated LOXL2 antibodies throughout their shelf life .

How should researchers interpret discrepancies between observed and calculated molecular weights for LOXL2?

Researchers frequently encounter differences between the calculated molecular weight of LOXL2 (87 kDa) and its observed molecular weight in experimental systems (100-105 kDa) . This discrepancy warrants careful interpretation:

• Post-translational modifications:

  • LOXL2 undergoes glycosylation which increases its apparent molecular weight

  • N-linked and O-linked glycosylation sites contribute to the higher observed mass

  • Treatment with glycosidases can confirm glycosylation as the source of molecular weight shift

  • Other modifications (phosphorylation, SUMOylation) may also affect migration

• Validation approaches:

  • Compare observed molecular weights across different cell types and tissues

  • Include recombinant LOXL2 (with known modifications) as a reference

  • Perform mass spectrometry analysis to accurately determine protein mass and modifications

  • Use multiple antibodies targeting different epitopes to confirm band identity

• Experimental considerations:

  • Gel percentage affects protein migration; lower percentage gels provide better resolution for higher molecular weight proteins

  • Running conditions (voltage, temperature) can influence apparent molecular weight

  • Different sample preparation methods may affect protein modifications

  • Variations in SDS-PAGE systems (Laemmli vs. Tris-tricine) may yield different apparent weights

• Multiple band interpretation:

  • Some LOXL2 antibodies detect multiple bands (e.g., 68 kDa and 100 kDa)

  • Lower molecular weight bands may represent:

    • Proteolytic fragments of LOXL2

    • Alternative splicing variants

    • Cross-reactivity with other LOXL family members

• Reporting considerations:

  • Always report both predicted and observed molecular weights

  • Document the experimental conditions that may affect migration

  • Include molecular weight markers in published western blot images

The consistent observation of LOXL2 at higher molecular weights across multiple studies with different antibodies supports that this represents the true physiological form of the protein rather than an artifact .

How are LOXL2 antibodies being utilized in hypoxia and exosome research?

Recent research has unveiled important connections between LOXL2, hypoxia, and exosome biology:

• Hypoxia-induced LOXL2 regulation:

  • LOXL2 expression and activity increases under hypoxic conditions

  • FITC-conjugated antibodies enable tracking of LOXL2 localization changes during hypoxia

  • Flow cytometry with FITC-LOXL2 antibodies allows quantification of expression changes in response to oxygen levels

• Exosome characterization:

  • LOXL2 has been identified as a component of exosomes, particularly from endothelial cells

  • Western blot analysis of exosomal fractions shows LOXL2 at approximately 105 kDa

  • Exosome-associated LOXL2 contributes to collagen crosslinking in recipient tissues

• Functional studies:

  • LOXL2 knockdown significantly reduces collagen crosslinking activity in hypoxic EC-derived exosomes

  • Exosomes from control endothelial cells induce collagen gel contraction, while exosomes from LOXL2 knockdown cells show reduced ability to promote contraction

  • Hypoxia enhances this exosome-mediated collagen crosslinking activity

• Methodological approaches:

  • Isolation of exosomes followed by Western blot detection of LOXL2

  • In vitro lysyl oxidase activity assays to measure functional LOXL2 in exosomes

  • Collagen gel contraction assays to assess the functional impact of exosomal LOXL2

  • Tracking of FITC-labeled antibodies to study LOXL2 transfer via exosomes

• Therapeutic implications:

  • Targeting exosomal LOXL2 may provide new approaches for fibrosis treatment

  • Monitoring LOXL2 in circulating exosomes could serve as a biomarker

  • FITC-conjugated antibodies facilitate screening of compounds that modulate LOXL2 exosomal packaging

These studies highlight the dynamic role of LOXL2 in cellular responses to hypoxia and intercellular communication via exosomes, opening new avenues for therapeutic intervention in fibrosis and cancer .

What are the current approaches for functional blocking studies using LOXL2 antibodies?

LOXL2 antibodies can be employed as functional blocking agents to study the enzyme's role in biological processes:

• In vitro enzymatic inhibition:

  • Direct measurement of lysyl oxidase activity in the presence of blocking antibodies

  • Dose-dependent inhibition curves to determine IC50 values

  • Comparison with small molecule inhibitors for mechanistic studies

  • Assessment of specificity through parallel inhibition of other LOX family members

• Cell-based functional assays:

  • Migration and invasion assays with antibody treatment

  • Collagen contraction assays to measure extracellular matrix remodeling

  • Cell adhesion studies to examine LOXL2's role in attachment to matrix proteins

  • EMT marker analysis following LOXL2 inhibition

• Recombinant antibody approaches:

  • Use of recombinant human IgG-based antibodies similar to simtuzumab

  • FITC conjugation to track antibody binding and internalization

  • Comparison of different epitope-targeting antibodies for optimal inhibition

  • Fc receptor-dependent versus independent effects assessment

• Experimental design considerations:

  • Time-course studies to determine optimal treatment duration

  • Pre-incubation versus co-incubation approaches

  • Controls including isotype-matched antibodies

  • Combination with genetic knockdown for validation

• Readout systems:

  • Western blot analysis of downstream signaling pathways

  • Immunofluorescence to monitor subcellular localization changes

  • qPCR to measure transcriptional effects of LOXL2 inhibition

  • Functional enzyme assays to directly measure LOXL2 activity inhibition

Researchers should note that functional blocking effects may be antibody clone-specific, with different epitope-targeting antibodies having distinct effects on LOXL2 enzymatic activity versus protein-protein interactions .

What are the emerging applications of FITC-conjugated LOXL2 antibodies in current research?

FITC-conjugated LOXL2 antibodies are finding expanding applications in cutting-edge research areas:

• Single-cell analysis:

  • Flow cytometry-based identification of LOXL2-expressing cells within heterogeneous populations

  • Cell sorting for downstream genomic or proteomic analysis

  • Correlation of LOXL2 expression with other surface and intracellular markers

  • Assessment of LOXL2 expression dynamics during cellular differentiation or disease progression

• Advanced imaging techniques:

  • Super-resolution microscopy for nanoscale localization of LOXL2

  • Live-cell imaging to track LOXL2 dynamics in real-time

  • FRET-based approaches to study LOXL2 interactions with binding partners

  • Tissue clearing methods combined with FITC-LOXL2 antibodies for 3D visualization

• Theranostic approaches:

  • Dual-purpose antibodies for both imaging and therapeutic applications

  • Monitoring therapeutic response using FITC-conjugated antibodies

  • Image-guided interventions targeting LOXL2-expressing tissues

  • Companion diagnostics development

• Microfluidic and organ-on-chip systems:

  • Real-time monitoring of LOXL2 in complex microenvironments

  • Assessment of LOXL2 expression in 3D culture systems

  • Evaluation of therapeutic interventions in physiologically relevant models

  • Studying LOXL2 in simulated pathological conditions (hypoxia, inflammation)

• Multi-parameter analysis:

  • Combination with other fluorescent markers for comprehensive phenotyping

  • Mass cytometry (CyTOF) incorporation for high-dimensional analysis

  • Spatial transcriptomics correlation with LOXL2 protein expression

  • Systems biology approaches to position LOXL2 in broader cellular networks

These emerging applications leverage the direct detection capabilities of FITC-conjugated LOXL2 antibodies to address increasingly sophisticated research questions about this important enzyme in health and disease .

How can researchers compare different LOXL2 antibodies to select the optimal reagent for their specific research question?

Systematic comparison of LOXL2 antibodies enables informed selection for specific research applications:

• Epitope mapping comparison:

  • Determine the target regions of different antibodies within the LOXL2 protein

  • Consider whether antibodies target the catalytic domain versus SRCR domains

  • Evaluate epitope conservation across species for cross-reactivity potential

  • Assess epitope accessibility in native versus denatured conditions

• Performance across applications:

  • Systematic comparison table:

• Validation evidence assessment:

  • Review published validation data using each antibody

  • Evaluate knockdown/knockout validation studies

  • Consider the extent of validation across different cell lines and tissues

  • Assess citation history in peer-reviewed literature

• Technical considerations for specific applications:

  • For multi-color flow cytometry: evaluate FITC-conjugated options

  • For co-localization studies: consider host species compatibility with other antibodies

  • For functional studies: prioritize antibodies with demonstrated blocking activity

  • For quantitative applications: select antibodies with linear detection ranges

• Practical implementation:

  • Conduct side-by-side testing of multiple antibodies on the same samples

  • Include appropriate positive controls (A549, HepG2, U-87 MG cells)

  • Evaluate lot-to-lot consistency through repeat testing

  • Document optimization parameters for selected antibodies