LAMA3 Antibody, Biotin conjugated

What is LAMA3 and what cellular functions do LAMA3 antibodies help investigate?

LAMA3 (Laminin Subunit Alpha 3) is a protein belonging to the laminin family of secreted molecules that are essential components of the extracellular matrix. Laminins are heterotrimeric molecules consisting of alpha, beta, and gamma subunits that assemble through a coiled-coil domain . LAMA3 functions primarily in:

• Mediating cellular attachment, migration, and tissue organization during embryonic development

• Facilitating cell adhesion via integrin alpha-3/beta-1 in focal adhesions and integrin alpha-6/beta-4 in hemidesmosomes

• Signal transduction via tyrosine phosphorylation of pp125-FAK and p80

• Differentiation of keratinocytes

LAMA3 antibodies enable researchers to investigate these biological processes through various techniques including immunohistochemistry, ELISA, and immunofluorescence. Biotin-conjugated LAMA3 antibodies are particularly valuable for enhancing detection sensitivity in complex tissue environments.

What are the optimal experimental conditions for using biotin-conjugated LAMA3 antibody?

Based on available product specifications, the optimal experimental conditions for biotin-conjugated LAMA3 antibody use include:

For immunohistochemistry of paraffin-embedded tissues (IHC-P), antigen retrieval is typically required. The antibody should be stored at -20°C in buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol . When working with the lyophilized format, reconstitution should follow the Certificate of Analysis instructions, generally using sterile PBS, pH 7.4 .

How can researchers validate the specificity of biotin-conjugated LAMA3 antibody?

Thorough validation of biotin-conjugated LAMA3 antibody is critical to ensure experimental reliability. Recommended validation approaches include:

• Positive and negative control tissues: Use tissues known to express LAMA3 (e.g., skin, kidney) as positive controls and tissues with minimal LAMA3 expression as negative controls

• Antibody specificity testing:

  • Western blot analysis to confirm reactivity with protein of the expected molecular weight (approximately 366.6 kDa for full-length LAMA3)

  • Dot blot analysis using the immunogen peptide or recombinant LAMA3 protein

• Knockdown/knockout validation: Compare staining patterns in LAMA3-silenced cells (using siRNA) to wild-type cells, as demonstrated in recent cholangiocarcinoma research

• Cross-reactivity assessment: Evaluate potential cross-reactivity with other laminin family members, particularly other alpha subunits

How can biotin-conjugated LAMA3 antibody be used to investigate LAMA3's role in cancer progression?

Recent research has demonstrated that LAMA3 plays significant roles in cancer progression, particularly in cholangiocarcinoma (CCA). Studies utilizing LAMA3 knockdown have revealed that:

• LAMA3 silencing reduced cell proliferation by up to 55% in KKU-213 cholangiocarcinoma cells

• LAMA3 knockdown significantly decreased cell adhesion, with only 36.0 ± 10.4% and 26.7 ± 9.7% of LAMA3-silenced HuCCA-1 and KKU-213 cells remaining attached compared to controls

• Cell migration was inhibited by 93% and 86% in HuCCA-1 and KKU-213 cells, respectively, following LAMA3 knockdown

Biotin-conjugated LAMA3 antibodies facilitate investigation of these processes through:

• Immunohistochemical profiling: Evaluating LAMA3 expression patterns in tumor versus normal tissue sections, utilizing the biotin-streptavidin system for signal amplification

• Co-localization studies: Examining LAMA3 interaction with focal adhesion proteins or integrin receptors through multi-color immunofluorescence

• Extracellular matrix (ECM) remodeling assessment: Analyzing changes in LAMA3 deposition during cancer progression and metastasis

• Therapeutic target validation: Confirming accessibility and specificity of LAMA3 as a potential therapeutic target in desmoplastic cancers

What methodological considerations are important when studying LAMA3 isoforms?

LAMA3 exists in multiple isoforms (up to 4 have been reported), making isoform-specific detection methodologically challenging . When using biotin-conjugated LAMA3 antibody to distinguish between isoforms, researchers should consider:

• Epitope mapping: Determine the specific region recognized by the antibody (e.g., the antibody described in result targets the region spanning amino acids 2701-2800 of the 3333 amino acid full-length LAMA3)

• Isoform-specific detection strategies:

  • Design experiments to differentiate between LAMA3A and LAMA3B variants

  • Consider that knockdown experiments may affect isoforms differently (e.g., siLAMA3 effectively inhibited LAMA3A expression by 73-86% but barely affected LAMA3B expression)

• Alternative approaches to complement antibody-based detection:

  • RT-PCR with isoform-specific primers

  • Mass spectrometry analysis for definitive isoform identification

  • RNA-seq analysis for transcriptomic profiling

How can biotin-conjugated LAMA3 antibody be integrated into multiplex immunoassays?

Multiplex detection systems represent an advanced application of biotin-conjugated LAMA3 antibody, enabling simultaneous analysis of multiple proteins within the same sample. Strategic approaches include:

• Sequential multiplexing with tyramide signal amplification (TSA):

  • Use biotin-conjugated LAMA3 antibody as the primary detection antibody

  • Apply streptavidin-HRP followed by tyramide-fluorophore for signal amplification

  • Strip or quench the signal before subsequent rounds of staining

  • This method allows detection of LAMA3 alongside other ECM components or integrins

• Complementary marker selection based on pathway analysis:

  • Include markers from enriched pathways identified in LAMA3-based studies

  • For example, LAMA3 knockdown affected SRC expression but not ITGA7, PIK3R1, or VASP

  • Consider focal adhesion pathway proteins as key complementary markers

• Technical considerations for reducing background:

  • Perform adequate blocking of endogenous biotin

  • Include appropriate controls for antibody cross-reactivity

  • Optimize detection systems to minimize autofluorescence

What are common challenges when using biotin-conjugated LAMA3 antibody and how can they be addressed?

Researchers may encounter several technical challenges when working with biotin-conjugated LAMA3 antibody:

• High background in biotin-rich tissues:

  • Problem: Endogenous biotin in tissues like liver, kidney, and brain can cause high background

  • Solution: Pre-block endogenous biotin using avidin/biotin blocking kits before antibody application

• Weak signal when detecting native LAMA3:

  • Problem: LAMA3 in the ECM may have limited accessibility due to protein-protein interactions

  • Solution: Optimize antigen retrieval methods (heat-induced epitope retrieval with citrate buffer at pH 6.0 or EDTA buffer at pH 9.0)

• Inconsistent staining patterns:

  • Problem: Variability in tissue fixation affecting epitope accessibility

  • Solution: Standardize fixation protocols and validate with positive control tissues known to express LAMA3

• Storage-related antibody degradation:

  • Problem: Loss of activity during storage or freeze-thaw cycles

  • Solution: Store at -20°C to -80°C for 12 months in lyophilized form; after reconstitution, aliquot and store at -80°C to avoid repeated freezing and thawing

How can researchers optimize detection sensitivity for low-abundance LAMA3?

When LAMA3 is expressed at low levels, several strategies can enhance detection sensitivity:

• Signal amplification systems:

  • Biotin-streptavidin-HRP system with tyramide amplification

  • Polymer-based detection systems that provide higher sensitivity than traditional ABC methods

• Sample preparation optimization:

  • Careful selection of fixation method and duration

  • Extended primary antibody incubation (overnight at 4°C)

  • Use of detection enhancers like protein crosslinkers

• Comparative analysis of detection methods:

  • Dot blot analysis may offer higher sensitivity for certain applications

  • Comparison of chromogenic versus fluorescent detection systems based on experimental needs

How is biotin-conjugated LAMA3 antibody used in studying epithelial integrity diseases?

LAMA3 is associated with several diseases affecting epithelial integrity, most notably Epidermolysis Bullosa, Junctional 2C, Laryngoonychocutaneous syndrome, and Epidermolysis Bullosa, Junctional 2A, Intermediate . Research applications of biotin-conjugated LAMA3 antibody in these disease models include:

• Basement membrane organization assessment:

  • Visualization of LAMA3 distribution at the dermal-epidermal junction

  • Quantification of LAMA3 expression in normal versus diseased tissue

  • Co-localization studies with other basement membrane components

• Mutation-specific expression analysis:

  • Characterization of LAMA3 expression patterns in tissues from patients with different LAMA3 mutations

  • Correlation of expression patterns with disease severity and clinical manifestations

• Therapeutic monitoring:

  • Evaluation of LAMA3 restoration following experimental therapies

  • Assessment of basement membrane reconstitution in response to treatment

What role does LAMA3 play in cancer research, and how can biotin-conjugated antibodies facilitate this research?

Recent studies have highlighted LAMA3's significance in cancer biology, particularly in cholangiocarcinoma. Biotin-conjugated LAMA3 antibodies contribute to cancer research through:

• Biomarker development:

  • LAMA3 has been identified as a potential diagnostic biomarker for cholangiocarcinoma

  • Immunohistochemical assessment of LAMA3 in tumor tissue microarrays for correlation with clinical outcomes

• Mechanistic studies of cancer progression:

  • Investigation of LAMA3's role in focal adhesion signaling

  • Analysis of LAMA3's contribution to epithelial-to-mesenchymal transition (EMT)

  • Studies have shown that LAMA3 knockdown affects expression of SRC, a key signaling molecule in cancer progression

• Therapeutic target validation:

  • Confirmation of LAMA3 accessibility in tumor tissues using biotin-conjugated antibodies

  • Evaluation of antibody penetration into desmoplastic stroma

  • Assessment of LAMA3 as a potential target for disrupting cancer cell-ECM interactions

How might biotin-conjugated LAMA3 antibodies contribute to emerging research in tissue engineering?

LAMA3's critical role in cell-ECM interactions positions it as an important molecule in tissue engineering applications. Biotin-conjugated LAMA3 antibodies can advance this field by:

• Biomimetic scaffold development:

  • Characterizing LAMA3 incorporation into engineered basement membrane constructs

  • Monitoring cellular response to LAMA3-containing matrices

  • Assessing stability and functionality of LAMA3 in artificial tissue environments

• Stem cell differentiation studies:

  • Evaluating LAMA3 expression during directed differentiation of stem cells

  • Correlating LAMA3 expression patterns with successful epithelial differentiation

  • Monitoring basement membrane formation in organoid cultures

• Integration with emerging technologies:

  • Combination with advanced imaging modalities for 3D visualization of LAMA3 distribution

  • Application in microfluidic systems to study dynamic cell-ECM interactions

  • Incorporation into high-throughput screening platforms for ECM-targeting therapeutics

What new methodological approaches might enhance LAMA3 antibody applications in research?

Emerging technologies offer opportunities to expand the utility of biotin-conjugated LAMA3 antibodies:

• Single-cell analysis integration:

  • Combining antibody-based detection with single-cell transcriptomics

  • Development of protocols for simultaneous protein and RNA analysis at the single-cell level

  • Correlation of LAMA3 protein expression with transcriptomic profiles

• Advanced imaging approaches:

  • Super-resolution microscopy for detailed visualization of LAMA3 organization

  • Expansion microscopy to enhance visualization of basement membrane architecture

  • Light-sheet microscopy for 3D visualization of LAMA3 in intact tissues

• Combinatorial analysis platforms:

  • Integration with mass cytometry for high-parameter analysis

  • Spatial transcriptomics combined with antibody-based protein detection

  • Development of computational tools for integrating multimodal data