Determination of transient or stable neo expression levels in mammalian cells.
We report an extension of the neomycin phosphotransferase II dot-blot assay to allow more rapid and sensitive quantitative determination of the neo gene product in crude mammalian cell extracts. Our procedure, based upon the dot-blot assay of Platt and Yang [Anal. Biochem. 162 (1987) 502-514], measures both the enzymatic activity and the protein content of a cell extract by scanning with an enzyme-linked immunosorbent assay reader, using the same sample rather than parallel samples for both measurements. We show this assay to be comparable to the chloramphenicol acetyltransferase assay in sensitivity. Therefore, apart from being a useful selectable marker gene, the neo gene is a convenient reporter gene in studies of stable, as well as transient, expression.
Duch
M
M
Department of Molecular Biology and Plant Physiology, University of Aarhus, Denmark.
Paludan
K
K
Pedersen
L
L
Jørgensen
P
P
Kjeldgaard
N O
NO
Pedersen
F S
FS
D000818:Animals; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D015971:Gene Expression Regulation, Enzymologic; D005819:Genetic Markers; D019868:Kanamycin Kinase; D010770:Phosphotransferases / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D005819:Genetic Markers; D015500:Chloramphenicol O-Acetyltransferase; D010770:Phosphotransferases; D019868:Kanamycin Kinase