A quantitative assay for neomycin phosphotransferase activity in plants.
A sensitive, simple, and quantitative assay for determining neomycin phosphotransferase (NPT) activity in plant cell extracts is described. The procedure retains the simplicity of previously published methods, yet offers up to a 140-fold increase in sensitivity. This increase is due to (1) the addition of bovine serum albumin (BSA) to the assay mixture, (2) desalting of crude maize extracts to remove a low-molecular-weight inhibitor of the enzyme, and (3) use of a different extraction buffer and an improved extraction procedure to liberate more enzyme from the cells. This method has been used successfully to detect and quantitate both stable and transient expression of NPT in transgenic tobacco and maize tissue.
Staebell
M
M
Pioneer Hi-Bred International, Inc., Department of Biotechnology Research, Johnston, Iowa 50313.
Tomes
D
D
Weissinger
A
A
Maddock
S
S
Marsh
W
W
Huffman
G
G
Bauer
R
R
Ross
M
M
Howard
J
J
D000818:Animals; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D005966:Glucuronidase / Q000378:metabolism; D006041:Goats; D007074:Immunoglobulin G / Q000494:pharmacology; D019868:Kanamycin Kinase; D008832:Microchemistry / Q000379:methods; D010047:Ovalbumin / Q000494:pharmacology; D010770:Phosphotransferases / Q000378:metabolism*; D010936:Plant Extracts / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D010947:Plants, Toxic; D011523:Protoplasts / Q000201:enzymology; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D014026:Nicotiana / Q000235:genetics; D003313:Zea mays / Q000235:genetics / Q000648:ultrastructure
D007074:Immunoglobulin G; D010936:Plant Extracts; D012710:Serum Albumin, Bovine; D010047:Ovalbumin; D010770:Phosphotransferases; D019868:Kanamycin Kinase; D005966:Glucuronidase