Coomassie blue stock

Stocks are analyzed by silver or Coomassie blue staining of capsid proteins separated on SDS polyacrylamide gels. As shown in Fig. 2.4A, the three capsid proteins (VP1, 2, and 3) are visible in the correct stoichiometry of 1 1 10, and are free of non-AAV proteins (>95% pure). 

Make up the volume with distilled water up to 1.6 ml. Then, add 0.4 ml of Coomassie Blue. Mix slowly, and measure the OD at 595 nm. Concentrations of the different enzyme dilutions can be determined using the standard plot. The concentration of -galactosidase stock solution is obtained by multiplying the enzyme concentration in the cuvette by the dilution factor. 

Bradford protein assay stock solution dissolve 150 mg of Coomassie Brilliant blue-G250 in 50 mL of ethanol (96%, HPLC grade). Mix extensively using a magnetic stirrer Filter the stock solution using Whatman No 1 filter paper and store the filtrate in the dark at 4°C. This solution is stable for several months (see Note 1). 

Protein reagent stock solution 0.05% (w/v) Coomassie brilliant blue G-250, 23.8% (v/v) ethanol, 42.5% (w/v) phosphoric acid. To make 200 ml of stock solution (5000 determinations), dissolve 0.1 g Serva blue G in 50 ml 95% ethanol (denatured ethanol usually works as well), add 100 ml 85% phosphoric acid, and make up to 200 ml by adding water. The stock solution is commercially available (Bio-Rad). Keep at 4°C. 

Protein determination measurements were performed by Bradford s Method. Bradford reagent was prepared by mixing 25 ml phosphoric acid, 12.5 ml ethanol and 25 mg Coomassie Brilliant Blue (G-dye). The mixture was diluted to 50 ml with distilled water. During measurements, a solution of Bradford reagent was prepared by mixing 1 volume stock solution with 4 volumes of distilled water. 

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