Optimization of purification procedure for potato virus Y strain NN

Potato virus Y strain NN (PVYNN) was purified from mechanically infected plants Nicotiana tabaccum cv. Samsun by extraction of the plants with various buffers, clarification of the suspensions with chloroform or Triton X-100, high speed centrifugation of the virus through sucrose cushion and resuspension of the sedimented virus is different buffers. The two optimal combinations of different procedures tested were either (1) extraction of the plants with the buffer containing 0.3% ascorbic, 0.3% mercaptoethanol, 0.01 mol/l diethyl pyrocarbonate (DEPC) and 5 mmol/l phenylmethylsulphonyl fluoride (PMSF), pH 5.3, clarification with cold chloroform, PEG precipitation, high speed centrifugation through sucrose cushion and resuspension of the sedimented virus in 0.02 mol/l Na-borate buffer pH 7.8, or (2) extraction of the plants with the buffer containing 0.5 mol/l Na-citrate, 1% mercaptoethanol and 5 mmol/l PMSF pH 6.5, clarification with 2% Triton X-100, PEG precipitation, high speed centrifugation through sucrose cushion and resuspension of the sedimented virus in 0.02 mol/l K-phosphate, 0.5 mol/l urea and 0.1% mercaptoethanol, pH 7.5.