Preparation of reagents Preparation of LB and Terrific broth Terrific broth 12 g of Pancreatic digest of casein 24 g of Yeast extract 9.

.4 g of Di-potassium phosphate 2.2 g of Mono-potassium phosphate In a total of 1000 mls Preparation of reagents used in PGP 1 expression

LB broth 10 g of Tryptone salt 5 g of Yeast extract 10 g of NaCl

1.0M IPTG stock Dissolved 1.9115g of IPTG powder in 5ml of distilled water; from the stock a working concentration of 1mM was used Transfer buffer 3.03 g Tris (25 mM Tris) 14.4 g Glycine (192 mM Glycine) 200 ml of methanol (20 % w/v) or without Final pH 8.3 to 1 liter Stacking buffer (0.5 M Tris-HCl (pH 6.8)) 6.0 g Tris base 60 ml of distilled water Adjusted pH to 6.8 with 6N HCl; made the mark to 100 ml with distilled water; stored at 4oC

Separating buffer (1.5 M Tris-HCl (pH 8.8)

27.23 g Tris base (18.15 g per 100 ml) 80 ml distilled water Adjusted pH to 8.8 with 6N HCl; made 150 ml with distilled water; stored at 4oC 10% SDS Desolved 10 g SDS powder in 90 ml of distilled water with gentle stirring and made to 100 ml with distilled water. Sample loading buffer Distilled water 0.5 M Tris-HCl (pH 6.8) Glycine 10% (w/v) SDS 2-mercaptoethanol 1% (w/v) bromophenol blue 5.8 ml 1.0 ml 0.8 ml 1.6 ml 0.4 ml 0.4 ml

Dilute 1:4 with sample buffer and heat at 95oC for 4 minutes 5X electrode buffer Tris base Glycine SDS 9 g (15 g/l) 43.2 g (72 g/l) 3 g (5 g/l)

Topped, 600 ml of water; stored at Rtp; for 1X, added 60 ml of 5X to 240 ml of distilled water Staining buffer (Coomasie) 0.1 % (w/v) coomasie blue 40% (v/v) methanol 10% (v/v) Glacial acetate Distilled water Distaining solution I 0.3 g 120 ml 30 ml 150 ml Distaining solution II

5% (v/v) methanol 7% (v/v) Acetic acid

40 % (v/v) methanol 10% (v/v) Acetic acid

Preparation of reagents used in Extraction and purification of PGP 1

Lysis buffer 50 mM Tris.Cl (pH 8.0) 1 mM EDTA 100 mM NaCl 8.0M Urea Dissolved 48.05g of Urea crystals in 100ml distilled water Urea buffer NaH2PO4.H2O Tris-base Urea 1.38g 0.12g 48.05g

Dissolved in 100ml distilled water; Adjust pH to 8.0 using NaOH

Wash buffer (used during protein elution from Ni-NTA column) 0.2% Tween 20 10% glycerol Urea buffer Imidazole mercaptoethanol Total volume is 5ml 10l 500l 4.44l 50l 0.7l

Elution buffer one (100mM) 0.2% Tween 20 10l

10% glycerol Urea buffer Imidazole mercaptoethanol Total volume is 5ml

500l 3.989l 500l 0.7l

Elution buffer two (200mM) 0.2% Tween 20 10% glycerol Urea buffer Imidazole mercaptoethanol Total volume is 5ml 10l 500l 3.4893l 1000l 0.7l

Elution buffer three (300mM) 0.2% Tween 20 10% glycerol Urea buffer Imidazole mercaptoethanol Total volume is 5ml Buffer A 50 mM Tris.Cl pH 8.0 5 mM EDTA 10 mM NaCl Buffer B 20 mM Na2HPO4 (pH 7.2) 20 mM NaCl 5mM EDTA 25% w/v sucrose 10l 500l 2.989l 1500l 0.7l