1.      Indirect ELISA protocol

1.1          Reagents：
    1.1.1     Coating Buffer: Carbonate Buffer (2.93 g NaHCO₃, 1.59 g Na₂CO₃, 1L ddH₂O)
    1.1.2     Diluent: PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5mM KH₂PO₄)
    1.1.3     Blocking Solution: 1% BSA in PBS
    1.1.4     Secondary Ab: anti mouse IgG-HRP(Sigma A2554-1ML)
    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 PBS
   2.1.4 Blocking Solution: 1% BSA in PBS
   2.1.5 Secondary Ab: anti mouse IgG-HRP(Sigma A2554-1ML) 
   2.2Assay 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.