Electrotransfer of [32P]NAD allows labeling of ADP-ribosylated proteins in intact Chinese hamster ovary cells
✍ Scribed by Michael C. Elia; Lawrence E. Motyka; Thomas D. Stamato
- Book ID
- 102987487
- Publisher
- Elsevier Science
- Year
- 1991
- Tongue
- English
- Weight
- 789 KB
- Volume
- 192
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
CHO-K1D cells electroporated in buffers containing [32P]NAD incorporated the label in a voltage-dependent manner. Electroporation with 650 V/cm at 1460 microF in Ham's F12 medium supplemented with 10 mM Hepes, pH 7.1, resulted in a greater than 20-fold increase in [32P]NAD uptake, while decreasing relative cellular survival by only 6%. Exposure of cells to gamma irradiation (20 Gy) prior to electroporation increased the steady-state level of poly(ADP-ribosylated) nuclear proteins two- to four-fold over that of unirradiated control cells. These data indicate that electrotransfer of [32P]NAD is a simple and rapid means of labeling the cellular NAD pool and should prove useful in the analysis of the relationship between poly(ADP-ribosylation) of nuclear proteins and DNA repair.