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.