4.6. Western Blotting

Fractions obtained after the anion exchange and gel filtration chromatography were tested for the presence of the toxin through Western blotting analyses. Protein content was determined by the Bradford method [23]. Proteins were separated on a 7% polyacrylamide gel and then electrically transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes with transferred proteins were incubated with the primary monoclonal antibody KT4 produced against a KT of W. anomalus ATCC 96603 and then with an anti-mouse secondary antibody. Protein detection was performed with an ECL Western blotting analysis system [24]. Each gel was loaded with pre-stained molecular weight markers in the range 10–245 kDa (Nippon Genetics, Düren, Germany).