33 

 PAGE 

 SDS gel electrophoresis 



SDS gel electrophoresis was prepared by following the 

 procedure of Laemmli (51) , with a 15% running gel and a 4% 

 stacking gel . A running buffer of Tris/glycine/SDS at pH 

 8.8 was used and the gel was run at constant voltage of 63 V 

 for about 8 h. The gel was stained for about 3 min in 

 Coomassie Brilliant Blue R250 dye, destained and viewed. 

 Silver staining 



Silver staining of an SDS gel as described by Pharmacia 

 (Piscataway, NJ) was followed. Washes of methanol/acetic 

 acid and ethanol/acetic acid were included before incubation 

 of the gel in oxidizer, followed by addition of the silver 

 stain, then developer, and finally an acetic acid/water wash 

 to stop the reaction. 

 Native gel electrophoresis 



A native PAGE was run according to the method of Shaw 

 and Prasad (74) . Gel concentrations were 7% for the 

 stacking gel and 15% for the running gel. The running gel 

 was pre-electrophoresed to remove the ammonium persulfate, 

 and fluorescent light was used to solidify the stacking gel 

 which contained riboflavin. The gel was run at constant 

 voltage of 60 V for about 8 h. 

 Shikimate dehydrogenase activity stained gel 



Optimal conditions for SDH activity stain on native 

 PAGE included 20 mg NADP\ 50 mg shikimate, 10 mg 3- (4,5- 



