**2.5. Ammonium sulfate precipitation of proteins**

The Crude (supernatant) was saturated to 45% (277g/l) by slowly adding ammonium sulfate salt at 40C for precipitation, kept it for overnight [5,6]. After precipitation the solution was centrifuged at 20,000g for 25 minutes [6,]. Collected the yellow supernatant saturated again to 95% by adding (NH4)2SO4 (372g/l) slowly and kept at 40C for overnight. The overnight precipitated solution was centrifuged at 23000g for 45 minutes. Pale supernatant was discarded. Precipitate (contains azurin) were collected and resuspended in 0.02M Potassium Phosphate buffer pH 7[11, 12].

## **2.6. Dialysis of the supernatant**

Azurin suspended in 0.02M potassium phosphate buffer pH 7 was dialysed by standard dialyses bag purchased from Sigma-Aldrich, (Kolkata, India) having 3 kDa molecular weight cut off at 40C for 20 hours on the same buffer for overnight with continuous gentle stirring. Dialysis was done until the solution attains its buffer pH. The solutions were kept at 40C after dialysis for further purification [11, 12].

## **2.6.1. Purification of Azurin on Ion – Exchange chromatography**

## *2.6.1.1. DEAE cellulose treatment*

Dialysate (contains azurin) were initially treated with DEAE. 100 ml slurry of DEAE cellulose equilibrated in 0.02M potassium phosphate buffer pH 7 were treated with the

dialysate and stirred for 20-30 min at 40C.The suspension was centrifuged at 10,000g for 15 min. Azurin does not adsorb in the gel remains in supernatant but most of the unwanted proteins like yellow flavo proteins are removed. Supernatant was collected. DEAE cellulose precipitate was resuspended in the same buffer and again centrifuged at 10,000g for 15 minutes to remove all unattached proteins [11-13].

Purification of Azurin from *Pseudomonas Aeuroginosa* 315

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

systems Illinois, USA) for analysis by using nitrogen laser at 337 nm.

*(MALDI-ToF)* 

*SDS-PAGE* 

purification process.

*2.6.6. FTIR analysis* 

transilluminator (Biorad, PA, USA) [16].

*2.6.4.1. Molecular weight determination by matrix-assisted laser desorption/ionization time of flight* 

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

*2.6.5. Purification profile of Azurin ((synthesized from P. aeruginosa MTCC2453) by* 

Five ml of 12% resolving gel contains 1ml distilled water, 30% acryl amide, 1.5M Tris (pH 8.8), 10% SDS, 10% APS and 0.002µl TEMED for polymerization was casted in the glass slab without any bubbles and kept it for 10-15 minutes. After polymerization of the resolving gel, 3ml of stacking gel (4%) were loaded over the resolving gel which contains 0.68 ml distilled water, 30% acryl amide, 1M Tris (pH 6.8) 10% SDS,10% APS, and 0.001ml TEMED. After casting the gel, proteins purified from different chromatography were loaded with bromophenol (molecular weight marker dye) at different lanes for profiling the protein

Glass slab gel were kept in the electrophoresis tank with tank buffer (196 mM glycine, 0.1%SDS, 50mM Tris-Hcl pH 8.3 made by diluting a 10x stock solution). This setup was connected with power pack initially in 80mV to 100 mV. After running the gel up to its anode end, was removed and stained with 0.2% coomassie brilliant blue for overnight. Destained with destaining solution (45: 45: 10 – methanol: water: acetic acid) which destains the comassie blue until it reveals the bands. The bands (figure 4) were observed under UV

Infrared spectroscopy experiments were performed using a Nexus 870 (Thermo Nicolet Corporation, Madison, USA) spectrometer equipped with a potassium bromide (KBr) beam splitter and DTGS (deuterated triglycine sulfate) detector in the range of 3,000-4000 cm−1. We recorded 32 scans per spectrum at a 2 cm−1 resolution for 100 µl of azurin liquid samples in 0.02 M PBS buffer (pH 7.0). We kept the same buffer as a background medium and performed all measurements at room temperature. We corrected spectra for the moisture

The supernatant after DEAE treatment was saturated to 100% (766g/l) with (NH4)2SO4 at 40C for overnight for precipitation. After saturation, precipitates are mixed gently and kept for centrifugation at 10000g for 10 min. supernatant was collected for dialysis at 40C for overnight with gentle stirring with the same before. Dialysis was continued till the solution pH attains its buffer pH [11-13].
