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Preparation of Reagents

The document describes the preparation of various reagents used for expression and purification of PGP1 protein, including: 1) Preparation of LB and terrific broth for bacterial growth. 2) Preparation of reagents for protein expression such as IPTG and sample loading buffer. 3) Preparation of lysis buffer, urea buffer, wash buffer, and elution buffers with varying imidazole concentrations for protein extraction and purification from Ni-NTA columns. 4) Preparation of electrophoresis buffers such as stacking buffer, separating buffer, and staining/destaining solutions.

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160 views4 pages

Preparation of Reagents

The document describes the preparation of various reagents used for expression and purification of PGP1 protein, including: 1) Preparation of LB and terrific broth for bacterial growth. 2) Preparation of reagents for protein expression such as IPTG and sample loading buffer. 3) Preparation of lysis buffer, urea buffer, wash buffer, and elution buffers with varying imidazole concentrations for protein extraction and purification from Ni-NTA columns. 4) Preparation of electrophoresis buffers such as stacking buffer, separating buffer, and staining/destaining solutions.

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kvicto
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© Attribution Non-Commercial (BY-NC)
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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

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