Quantification of ADP-ribosylated Actin

To quantify the amount of toxin-induced ADP-ribosylation of actin in intact cells, the indirect method of differential ADP-ribosylation is appropriate. Assuming that in intact cells C2 modifies cellular actin, subsequent pP]ADP-ribosylation of the lysates should result in a decreased incorporation of pP]ADP-ribose. Because C2I shows transferase activity on ice, although less than at 37°C, the cells should be lysed in a buffer containing [ P]NAD and C2I. 

Cultured cells are rinsed with ice-cold PBS and were then mechanically removed in the presence of 1 mM MgCl2, 50 mM HEPES pH 7.5, 0.3 mM phenylmethylsulfonly fluoride, 30 ig/ml leupeptine, 1 mM dithiothreitol, 10 mM thymidine, 5 M [ P]NAD, 

The ADP-ribosylation reaction is stopped by addition of Laemmli sample buffer, or the proteins are precipitated with 1 ml of trichloroacetic acid (20%, w/v). 

Probing the Actin Cytoskeleton by Clostridium botulinum C2 Toxin and Clostridium perfringens lota Toxin 133 

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