In situ electroporation of radioactive compounds into adherent cells.
We previously developed a technique, termed in situ electroporation, where nonpermeant molecules are introduced through an electrical pulse into adherent cells, while they grow on electrically conductive, optically transparent, indium-tin oxide (ITO). Careful control of the electric field intensity results in essentially 100% of the cells taking up the introduced material, without any detectable effect upon the physiology of the cell, presumably because the pores reseal rapidly so that the cellular interior is restored to its original state. Electroporation of radioactive material is faced with two important considerations: (1) potential for exposure of personnel to irradiation, and (2) the requirement for electroporation of a large number of cells. In this report, we describe a modification in the geometry of the slides and electrodes which permits the use of inexpensive ITO-coated glass of lower conductivity that can be discarded after use, to electroporate large numbers of cells using a minimum volume of radioactive nucleotide solution. The results demonstrate that, using this assembly, the determination of the Ras-bound GTP/GTP+GDP ratios through electroporation of [alpha32P]GTP can be conducted using approximately five times lower amounts of isotope than in previous designs. Moreover, this assembly permits efficient upscaling, which makes the determination of Ras-GTP binding in cells which are deficient in Ras activity possible. In addition, we demonstrate the labeling of two viral phosphoproteins--the Simian Virus 40 Large Tumor antigen, and Adenovirus E1A--through [gamma32P]ATP electroporation using this setup. In both cases, electroporation of the nucleotide can achieve a great increase in the efficiency and specificity of labeling compared to the addition of [32P]-orthophosphate to the culture medium, presumably because the immediate phosphate donor nucleotide itself is introduced, which can directly bind to the target proteins.
Tomai
Evangelia
E
Department of Microbiology, Queen's University, Kingston, Ontario, Canada.
Vultur
Adina
A
Balboa
Valerie
V
Hsu
Tina
T
Brownell
Heather L
HL
Firth
Kevin L
KL
Raptis
Leda
L
D017500:Adenovirus E1A Proteins / Q000378:metabolism*; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D018274:Electroporation / Q000379:methods; D005347:Fibroblasts / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D051379:Mice; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D010761:Phosphorus Radioisotopes / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism
D017500:Adenovirus E1A Proteins; D000952:Antigens, Polyomavirus Transforming; D010761:Phosphorus Radioisotopes; D014764:Viral Proteins; D006153:Guanosine Diphosphate; C077418:L protein, Simian parainfluenza virus 41; D006160:Guanosine Triphosphate; D012321:DNA-Directed RNA Polymerases; D015689:Oncogene Protein p21(ras)