*2.6.3. Purification of Azurin on ion – Exchange (anionic) chromatography*

## *2.6.3.1. Chromatography on CM cellulose*

The CM cellulose beads from Sigma–Aldrich (Kolkata, India) were equilibrated for overnight in the ammonium acetate buffer pH 3.9 adjusting the pH by 0.05M acetic acid with 2M NH3.After swelling, the beads were packed in a 5cm x 15cm glass column and washed for ten times of the column volume. Gently one ml of the sample added (Fraction (e) collected from G-75) over the top of the column and left it for 5-10 minutes to bind the protein inside the beads. After 10 minutes the column was eluted with ammonium acetate buffer pH 4.65 [12-14].

## *2.6.4. Characterization of Azurin (purified from P. aeruginosa MTCC2453)*

## *2.6.4.1. Molecular weight determination by matrix-assisted laser desorption/ionization time of flight (MALDI-ToF)*

The most successful method to analyze biopolymers such as, proteins, peptides, sugars and large organic molecules which are tend to be fragile and fragment when ionized by more conventional ionization methods [15]. The Fractions collected from G-25, G-75 and CM cellulose were performed MALDI for molecular weight determination. Two micro liter from each fraction of the chromatography was added with 20µl of 3, 5-Dimethoxy, 4-Hydroxy cinnamic acid otherwise called as sinnapinic acid (Sigma-Aldrich. Kolkata, India). Tiny spots were made on silver plate and kept for drying for 4-6 hours to drain the water molecules. Further spots were dried with a vacuum drier to make a crystalline molecule. After drying the samples were placed in the MALDI-ToF chamber (Voyager De pro, applied systems Illinois, USA) for analysis by using nitrogen laser at 337 nm.
