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.
A new protein-free blocking system containing 10% (w/v) sulfanilic acid/10% (v/v) triethanolamine/0.05% (v/v) Tween 20 has been used to block free binding sites of the covalently binding matrix ...cyanuric chloride-activated paper (CCA-paper). This method allows a reversible staining of protein blots with Coomassie brilliant blue after each step of the immunological procedure and reuse of the blots for a repeated antibody probing. Non-radioactive and radioactive detection procedures have been compared with blots on CCA-paper and nitrocellulose. The best combination is a Coomassie brilliant blue staining and immunological detection with 125I-protein A.
