Galectin-4(GAL4) is a protein that in humans is encoded by the LGALS4 gene. The galectins are a family of beta-galactoside-binding proteins implicated in modulating cell-cell and cell-matrix interactions. LGALS4 is an S-type lectin that is strongly underexpressed in colorectal cancer. In this case, we chose rabbit erythrocyte (RaE) to assay its ability of agglutination. A general procedure for hemagglutination assay (or haemagglutination assay; HA) is as follows, two-fold dilute the recombinant cattle GAL4 with 0.9% sodium chloride injection, add 50μL a serial dilution of GAL4 to each well of a U or V-bottom shaped 96-well microtiter plate. The final well serves as a negative control without GAL4, replace with 50μL 0.9% sodium chloride injection. Then add 50μL 1% rabbit erythrocyte to each well and mixed gently. The plate is incubated for 3 hours at room temperature. The results are shown in Figure 1. It was obvious that the minimal effective concentration of GAL4 is 3.1 μg/mL.

Figure 2. The hemagglutination assay of GAL4 in V- bottom shaped 96-well microtiter plate.