35 ml 0 6 mg of proteinase k dissolved in 1 36 ml of 20 mm cacl 2 aqueous solution was added the mixture was incubated at 37 u00b0 c and after 2 5 hours placed in an ice water bath a sample was taken and analyzed by sds page sample in lane 2 fig1 impurities in this sample represented about 14 of the sample weight fifteen minutes afterwards about 50 u03bcl of 10 acetic acid aqueous solution was added in an ice water bath to bring the ph of the liquid to 4 1 then at the same temperature and with vigorous agitation 1 54 ml of ethanol was slowly added the liquid was kept at 0 u00b0 c for 30 minutes and centrifuged at 16 000 g at 4 u00b0 c for 20 min the pellet was suspended in 2 ml of an aqueous buffer consisting of 20 mm tris hcl and 10 mm mgcl 2 adjusted to ph 7 5 a sample was taken and analyzed by sds page sample in lane 3 fig1 impurities in this sample represented about 10 of the sample weight the diluted sample was filtered through a vivaspin 2 sartorius 100 kda membrane from which 200 u03bcl of retentate was obtained the retentate was then diluted to 2 ml with the same buffer and filtered through the same 100 kda membrane dilution and ultrafiltration of the retentate 200 u03bcl was repeated four more times a.