Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay.
Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells. We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene. The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene neo; hph and neo may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.
Sørensen
M S
MS
Department of Molecular Biology, University of Aarhus, Denmark.
Duch
M
M
Paludan
K
K
Jørgensen
P
P
Pedersen
F S
FS
D000818:Animals; D002457:Cell Extracts; D002460:Cell Line; D005819:Genetic Markers; D019868:Kanamycin Kinase; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D013379:Substrate Specificity
D002457:Cell Extracts; D005819:Genetic Markers; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D019868:Kanamycin Kinase