KOSEN 한인과학기술자네트워크

논문에 나온 방법입니다. Eukaryot Cell. 2003 October; 2(5): 886–900. Chitin Synthesis in Saccharomyces cerevisiae in Response to Supplementation of Growth Medium with Glucosamine and Cell Wall Stress Dorota A. Bulik, Mariusz Olczak, Hector A. Lucero, Barbara C. Osmond,1 Phillips W. Robbins, and Charles A. Specht Measurement of the chitin content of cells. The Morgan-Elson method (29) for colorimetric determination of GlcNAc was adapted for microplate readers in measurements of cellular chitin levels. Cultures for chitin determination were made from those initially grown to stationary phase in liquid YPD medium and then diluted 1:100 in fresh medium and incubated at 30°C with shaking for 18 to 22 h. Typically, 1 ml of culture was spun in a tared microcentrifuge tube and then washed once with 1 ml of water, and all residual liquid was removed from the pellet to yield 10 to 25 mg (wet weight) of cells. The cells were suspended in 0.5 ml of 6% KOH and heated at 80°C for 90 min. Samples were centrifuged at 20,000 × g for 10 min, and the supernatant was discarded. The pellet was suspended in 1 ml of phosphate-buffered saline and spun again, and the buffer was discarded. Each pellet was suspended in 0.1 ml of McIlvaine's buffer, pH 6.0, and 5 μl of purified Streptomyces plicatus chitinase-63 (5 mg/ml in phosphate-buffered saline) was added to hydrolyze chitin to GlcNAc; samples were incubated for 24 h at 37°C. Ten microliters of 0.27 M sodium borate, pH 9.0, and 10 μl of sample supernatant were combined in 0.2-ml PCR tubes. Samples were heated in a thermocycler (Techne Inc., Princeton, N.J.) to 99.9°C for about 60 s, mixed gently, and incubated further at 99.9°C for 10 min. Immediately upon cooling to room temperature, 100 μl of freshly diluted DMAB solution (Ehrlich's reagent, consisting of 10 g of p-dimethylaminobenzaldehyde in 12.5 ml of concentrated HCl and 87.5 ml of glacial acetic acid, diluted 1:10 with glacial acetic acid) was added to samples, which were incubated at 37°C for 20 min. Seventy-five microliters was transferred to 96-well low-evaporation microtiter plates, and absorbance at 585 nm was recorded. Standard curves were prepared from stocks of 0.2 to 2.0 mM GlcNAc. 그리고 아래의 논문에도 Cell Wall의 Chitin의 농도를 측정하는 방법이 쓰여 있으니 참고하셔도 될 수 있습니다. Independent regulation of chitin synthase and chitinase activity in Candida albicans... Selvaggini et al. Microbiology.2004; 150: 921-928. > >미생물을 구성하는 키틴의 함량이나 비율을 알수 있는 실험방법 > >알고 계시면 자료나 방법좀 알려주시면 고맙겠습니다.

chitin과 chitosan의 비율을 알고 싶은신가요, 아님 전체 미생물의 다당체중에서 chitin의 비율을 알고 싶으신가요? >미생물을 구성하는 키틴의 함량이나 비율을 알수 있는 실험방법 > >알고 계시면 자료나 방법좀 알려주시면 고맙겠습니다.

제가 미생물을 배양해서 그것의 균체를 얻었을때.. 미생물내에 있는 키틴의 함량을 알고 싶습니다. 미생물에서 키틴이 얼마만큼 차지하고 있는가를 알고 싶은데요.. 미생물에 포함되어 있는 키틴의 함량을 알고 싶습니다.