반응 #705811
ord-7fc462efd09f4b889f1ff520e1d52a35
반응 조건
후처리
- 1기타were prepared
- 2추출extract
- 3workup.ADDITIONAcceptor substrates were added to a final concentration of 20 mM
- 4기타terminated by addition of 20 μl ethanol
- 5workup.ADDITIONfollowed by addition of 600 μl of distilled water
- 6기타An aliquot of each reaction (50 μl)
- 7기타in the reaction
- 8세척The flow through fraction, and 2 μl of a subsequent water elution
- 9기타were collected
실험 절차
Cell extracts containing 1% Triton X-100 were prepared from transfected COS-1 cells. Fucosyltransferase assays were performed in a total volume of 20 μl, and contained 50 mM sodium cacodylate, pH 6.2, 5 mM ATP, 10 mM fucose, 20 mM MnCl2, 3 μM GDP-14C-fucose, and 5 μl (30 μg protein) of cell extract. Acceptor substrates were added to a final concentration of 20 mM. Reactions were incubated at 37° C. for 1 hour and terminated by addition of 20 μl ethanol, followed by addition of 600 μl of distilled water. An aliquot of each reaction (50 μl) was subjected to scintillation counting to determine total radioactivity in the reaction. Another aliquot (200 μl) was applied to a column containing 400 μl of Dowex IX2-400, formate form. The flow through fraction, and 2 μl of a subsequent water elution, were collected and pooled, and an aliquot was subjected to scintillation counting to quantitate incorporation of radioactive fucose into neutral product. Descending paper chromatography was used to confirm the structure of the product formed with the acceptor N-acetyllactosamine. The neutral product in the Dowex column eluate was concentrated by lyophilization, resuspended in a small volume of water, and fractionated through Whatman No. 40 in phenol/isopropanol/formic acid/water (85:5:10:100, lower layer). After chromatography (40 hours), the air-dried chromatogram was cut into 1 cm strips and the strips eluted into 2 ml of water. Radioactivity in each eluate was determined by scintillation counting after mixing with 10 ml of scintillation cocktail.