Specification
Product name             Galectin-3 Blocking Antibody 5-8
Description                 Mouse monoclonal antibody raised against a   full-length recombinant Galectin-3.
                                   Monoclonal antibodies block the binding of Galectin-3 to laminin.
Immunogen               Recombinant full length protein. This information is proprietary to RMi and/or its  
                                   suppliers
Source/Isotype          Mouse IgG1
Reactivity                   Human
Specificity                  Detects human Galectin-3 in ELISAs. In indirect ELISAs, no cross-reactivity with
                                   recombinant human Galectin-1, Galectin-9 is observed.
Purification                Protein G purified from hybridoma culture supernatant
Application                 ELISA, others unspecified.
Form                           Liquid
Storage Buffer           In 1✕PBS, pH 7.2

Storage Instruction   Shipped at 4°C. Store at +4°C short term (1-2 weeks). Store at -20°C or lower long
                                   term.Aliquot to avoid repeated freezing and thawing.
Size                            25ug, 100ug, 500ug

Data Background

The galectins constitute a large family of carbohydrate-binding proteins with specificity for N-acetyl-lactosamine-containing glycoproteins. At least 14 mammalian galectins, which share structural similarities in their carbohydrate recognition domains (CRD), have been identified. The galectins have been classified into the prototype galectins (-1, -2, -5, -7, -10, -11, -13, -14), which contain one CRD and exist either as a monomer or a noncovalent homodimer; the chimera galectins (Galectin-3) containing one CRD linked to a nonlectin domain; and the tandem-repeat galectins (-4, -6, -8, -9, -12) consisting of two CRDs joined by a linker peptide. Members of the galectin family have been implicated in a variety of biological functions including cell adhesion, growth regulation, cytokine production, T-cell apoptosis, and immune responses. Galectin-3 is involved in several diverse biological functions. Galectin-3 binds IgE. Galectin-3 is an unusual protein in that it can be found both extracellularly and intracellularly. Intracellularly, galectin-3 can localize to the cytoplasm, nucleus, or both, depending on cell type and experimental conditions. Nuclear galectin-3 has been identified as a pre-mRNA splicing factor. Galectin-3 production has been shown to increase during inflammation and in obesity, and the protein itself can have an inflammatory effect under certain conditions. Galectin-3 forms a complex with α3, β1 integrin to act as a surface receptor on endothelial cells for the NG2 proteoglycan, which triggers cell motility and angiogenesis. In addition to these functions, galectin-3 is also a required factor for the terminal differentiation of epithelial cells. 

1. Indirect ELISA protocol

1.1   Reagents：

1.2    Assay Protocol：

       Note: The following protocol is a guideline, user need to determine their optimal experimental condition for best performance.

1.2.1 Coat plate with Recombinant protein 0.2ug/ml, 100ul/well, incubate at 4℃ for overnight.

1.2.2 Wash the plate with PBST (0.05% Tween-20) once.
1.2.3 Add 200μL of blocking buffer each well, incubate the plate at 37℃ for 2hr or 4℃ overnight.
1.2.4 Add sample 100ul/well, incubate the plate at 37℃ for 1hr.
1.2.5 Wash the plate with PBST (0.05% Tween-20) 3 times.
1.2.6 Add secondary Ab, 1:10k diluted anti-mouse IgG-HRP, 100ul/well, incubate the plate at 37℃ for 0.5hr.
1.2.7 Wash the plate with PBST (0.05% Tween-20) 3 times.
1.2.8 Add 100ul/well TMB, incubate at room temperature for 10min, add 50ul/well 1N HCl or 2% H2SO4.
1.2.9 Read absorbance at 450nm.

2.      Galectin-3 Antibody binding/blocking assay protocol
2.1   Reagents：

2.1.1     Coating Buffer: PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5mM KH₂PO₄)

2.1.2     Laminin: Corning, 354232

2.1.3     Diluent: 1% BSA in PBS21.1.4     Blocking Solution: 1% BSA in PBS

2.1.5     Secondary Ab: anti mouse IgG-HRP(Sigma A2554-1ML)

 2.2  Assay Protocol：

Note: The following protocol is a guideline, user need to determine their optimal experimental condition for best performance.

2.2.1     Coat plate with Laminin 5ug/ml, 100ul/well, Incubate at 4℃ for overnight.

2.2.2     Wash the plate with PBST (0.05% Tween-20) once.

2.2.3     Add 200μL of blocking buffer each well, incubate the plate at 37℃ for 2hr or 4℃ overnight.

2.2.4     Add the plate with a series of anti-Galectin-3-Ab 50ul/well, and then add 2.0ug/ml Galectin-3 solution 50ul/well, incubated at 37℃ for 1hr.

2.2.5     Wash the plate with PBST (0.05% Tween-20) 3 times

2.2.6     Add secondary Ab, 1:10k diluted anti-mouse IgG-HRP, 100ul/well, incubate the plate at 37℃ for 0.5hr.

2.2.7     Wash the plate with PBST (0.05% Tween-20) 3 times

2.2.8     Add 100ul/well TMB, incubate at room temperature for 10min, add 50ul/well 1N HCl or 2% H₂SO₄.

2.2.9     Read absorbance at 450nm.