transferred to nitrocellulose sheets using a semi dry electroblotter integrated separation systems hyde park mass and then stained for the presence of bont a specific protein using horse antiserum to bont a strain mtd150 was prepared as described above and used as a background control purified bont a sigma st louis mo was included in each gel as a positive control cell fractionation bacterial strains encoding the truncated bont a proteins were grown in m 9 medium and were induced to express protein as described above cells were pelleted by centrifugation at 3 000 u00d7 g for 10 min the pellet was suspended in lysis buffer containing 1 mg ml lysozyme 50 mm tris 50 mm edta and 20 sucrose ph 8 0 and was incubated at 37 u00b0 c for 30 min to ensure complete lysis the cell suspension was subjected to two cycles of rapid freeze thaw mgso 4 was added to the lysate to a concentration of 20 mm dnase sigma and rnase sigma were added to a concentration of 0 01 mg ml each and then the lysate was incubated at 37 u00b0 c for 30 min the lysate was clarified by centrifugation at 3 000 u00d7 g for 10 min the clarified lysate was centrifuged at 20 000 u00d7 g for 30 min at 4 u00b0 c and the resulting pellet was dissolved in sample loading buffer the sample was boiled for 5 min and stored at u2212 20 u00b0 c before use purification of bont a proteins the truncated bont a proteins were purified by preparative sds page with a model 491 prep cell bio rad richmond calif the percentage of acrylamide used in the resolving gel was adjusted to maximize the separation of the protein of interest separation was typically performed at 12.