ADP-ribosylation of Actin in Cell Lysates

ADP-ribosylation of actin in cell lysates is performed in the following way  

40jil of cell lysate plus 10 xM of pPJNAD (0.3fiCi), plus 10 mM thymidine and 1 mM dithiothreitol is incubated with 0.1 [j,g/ml of iota toxin (total volume 50. 1) for 30 min at 37°C. Thymidine blocks the poly(ADP-ribose)polymerase (approx. 120 kDa), thereby preventing consumption of NAD, which is essential for quantitative ADP-ribosylation of actin. 

The ADP-ribosylation reaction is terminated by addition of 10 n.1 Laemmli sample buffer, or by precipitation with 1 ml of trichloroacetic acid (20 %, w/v). 

The proteins are separated by 11 % SDS-gel electrophoresis, and labeled actin is analyzed by phosphorimaging or autoradiography. 

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