Coomassie brilliant blue G250 色素結合法による高感度タンパク質定量法

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  • Coomassie brilliant blue G250 dye-binding microassay for protein

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The high sensitive reagent using Coomassie brilliant blue G250 (CBB) for protein microassay has been prepared. At first, the high sensitive coloration system (0.1mL protein solution and 1mL Bradford reagent) was examined with Bradford reagent (0.01%(w/v) CBB-4.75%(v/v) ethanol-8.5%(w/v) phosphoric acid) according to Bradford (1976) using BSA(0-9μg) as a standard. The Bradford reagent produced CBB precipitate just after preparation and during storage, so filtration of it was needed before every use. Therefore, calibration of the Bradford CBB reagent due to change in CBB concentration should be carried out, which required time. These demerits had not been overcome by our modifications of the Bradford reagent. Thus we developed a stable, sufficiently available CBB reagent without CBB precipitate during storage. The CBB reagent is the 0.025%(w/v) CBB-12.5%(v/v) methanol-70.83%(w/v) phosphoric acid. The protein coloration with CBB was done at 0.8mL protein solution(0-9μg protein) and 0.2mL CBB reagent.

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