Assay of neomycin phosphotransferase activity in cell extracts.
A simple method for the measurement of neomycin phosphotransferase (NPT) activity in crude extracts of eukaryotic cells is described. This method is based on the elimination of interfering phosphorylated proteins by using phenol-chloroform extraction. This solution phase assay allows the detection of greater than or equal to 0.01 ng of NPT in the crude cell extract. Rapid screening of a large number of cell cultures generated in gene-transfer experiments, using NPT as a selective marker, is made possible by this simple technique. Further, the promoter strength of vector constructs used in gene therapy may also be estimated by this procedure.
Ramesh
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Department of Pediatrics, University of Washington, Seattle 98195.
Osborne
W R
WR
D002457:Cell Extracts / Q000032:analysis*; D002460:Cell Line; D006801:Humans; D019868:Kanamycin Kinase; D010770:Phosphotransferases / Q000032:analysis*; D015203:Reproducibility of Results
D002457:Cell Extracts; D010770:Phosphotransferases; D019868:Kanamycin Kinase