Identification of haemagglutinating fractions obtained by anion exchange chromatography.
A. Ion exchange chromatogram of protein extract (PE) of E. tirucalli latex: the eluted fractions were collected (1 mL each, in a flow of 2 mL.min−1) and monitored spectrophotometrically in A280nm. All fractions obtained were tested as regards the haemagglutinating activity. Peak a corresponds to the non-interacted fraction; b and c represent proteins that interacted with the column. B. SDS-PAGE (12,5%) crude protein latex extract (PE) and fractions obtained by chromatography (1, 7, 10, 11, 12, 13, 14, 15 and 16). Closed triangles (▴) represent the haemagglutination observed after membrane washing with TBS. Mr, correspond to the molecular pattern, whose numbers on the left indicate the mass values. C. Determination of haemagglutinating potential of fractions by dot-blot: 200 µL aliquots of each fraction were applied on nitrocellulose membrane, and later incubated with A+ human erythrocytes (2%) for 30 min. D. Relative quantification of the 32KDa protein band and agglutinated eritrocites band on the nitrocellulose support by densitometric analysis using ImageMaster 2D Platinum 7.0. Vertical bars correspond to the standard deviation of the average of three replicates. Different letters indicate significant statistical difference between samples by the Tukey test (p<0,05).