slow stiffing the buffer was changed at the ratio of 1 10 for another three hours dialysis with the same slow stirring for proteins tagged with 6 his 10 mm imidazole was added to the dialysate and loaded on 5 ml histrap hp u00ae column sigma aldrich precharged with nickel and pre equilibrated with 25 mm tris ph8 0 0 15m nacl 0 15 n lauroylsarcosine the column was washed with 50 ml washing buffer 25 mm tris ph8 0 0 15m nacl 0 15 n lauroylsarcosine 10 mm imidazole the bound proteins were eluted with elution buffer 25 mm tris ph8 0 0 15m nacl 0 15 n lauroylsarcosine 0 5m imidazole for proteins not labeled with 6 his the dialysate was loaded on a 5 ml hitrap q u00ae column sigma aldrich pre equilibrated with loading buffer 20 mm tris ph8 0 50 mm nacl 0 15 n lauroylsarcosine 0 5 mm edta 1 mm pmsf the column was washed with 50 ml washing buffer 20 mm tris ph8 0 150 mm nacl 0 15 n lauroylsarcosine 0 5 mm edta 1 mm pmsf the protein was eluted with linear gradient nacl from 150 mm to 725 mm in 20 mm tris ph8 0 0 15 n lauroylsarcosine 0 5 mm edta 1 mm pmsf for increased purity the protein containing fractions were concentrated and further separated with a superose u00ae 6 10 30 column amersham biosciences the running buffer was 20 mm tris ph8 0 150 mm nacl 0 15 n lauroylsarcosine 0 5 mm edta 1 mm dtt kaur et al u201c purification and characterization of aminopeptidase n from spodoptere litura.