*2.6.2. Purification of Azurin on gel-filtration chromatography*

## *2.6.2.1. Chromatography on Sephadex G-25*

Sephadex G-25 beads were equilibrated in the 0.02M potassium phosphate buffer pH 7 [Parr S R et al 1976] for overnight, and tightly packed in 3cm x 25cm length glass column without any bubbles. The column was initially washed with 0.02M potassium phosphate buffer for twenty volumes of the gel packed. The Flow rate was adjusted to one minute per ml. slowly the dialysate (after DEAE treatment) was added with the eluent buffer 0.02M potassium phosphate buffer pH 7 on the column. Thirty fractions were collected at one minute interval [12-14].

## *2.6.2.2. Chromatography on Sephadex G-75*

Sephadex G-75 beads in powder form are equilibrated in 0.01M Tris/Hcl buffer pH 7.5 for overnight. After equilibration the beads were tightly packed in a 3cm x 45cm glass column. The column was washed with same equilibrating buffer for fifty volumes of the column value. After washing with buffer one ml of the sample (fraction (a) collected from the G-25) were passaged and eluted with the same equilibrating buffer. Seventy five fractions were collected at 1 ml/6minutes flow rate [12-14].
