A new neomycin phosphotransferase II solid phase assay in combination with polyacrylamide sodium dodecylsulphate gel electrophoresis.
A new general method for the determination of neomycin phosphotransferase (NPT) II (EC 2.7.1.95) activity in cell extracts after separation in SDS-polyacrylamide gels is described. The enzymatic activity of NPT II is restored after SDS-polyacrylamide gel electrophoresis by incubating the gel for 3 h (20 mM Tris-HCl buffer, pH 7.4). The enzymatic activity is determined by in situ phosphorylation of aminoglycoside antibiotics bound to solid supports and brought into direct contact with the gel surface. A novel, mechanically stable, negatively charged matrix was synthesized for use in this solid phase enzyme assay and compared to phosphocellulose and carboxymethylcellulose paper. This new method allows the easy and exact determination of the molecular weight of any fusion protein with NPT II by assaying the position of the enzymatic activity in the gel and a consecutive immunological reaction following protein transfer onto nitrocellulose membranes.
Merkel
D
D
Academy of Sciences of the G.D.R., Central Institute of Molecular Biology, Department of Human Molecular Genetics, Berlin, G.D.R.
Schmidt
G
G
Flachmeier
C
C
Behrendt
G
G
Coutelle
C
C
Hunger
H D
HD
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001345:Autoradiography; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D019868:Kanamycin Kinase; D008970:Molecular Weight; D010770:Phosphotransferases / Q000032:analysis*; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012967:Sodium Dodecyl Sulfate
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D011993:Recombinant Fusion Proteins; D012967:Sodium Dodecyl Sulfate; D010770:Phosphotransferases; D019868:Kanamycin Kinase