G. α-Gal Quantitative Assay.doc

G. α-Gal Quantitative Assay G. α-Gal定分析 Reagents and Materials Required: 材料与试剂： ? Appropriate liquid synthetic dropout (SD) culture medium (Appendix C.A) 适量SD液体培养基 ? 50-ml culture tubes 50ml 培养管 ? PNP-α-Gal Solution (100 mM) 100 mM PNP-α-Gal溶液 ? 10X Stop Solution (Appendix D.F) 10X 终止液 （1 M Na2CO3溶液） ? 1X NaOAc Buffer (Appendix D.F) 0.5M 醋酸钠 ? Assay Buffer (Appendix D.F) 分析缓冲液：现配 0.5M 醋酸钠溶液与100 mM PNP-α-Gal溶液按体积比2:1混合 ? 1.5-ml cuvettes or 96-well, flat-bottom microtiter plates for OD410 measurements 1.5ml比色杯或96孔板，OD410 酶标仪 Preparation of Samples 样品准备： 1. Inoculate 2–5 ml of liquid synthetic dropout (SD) medium, containing the appropriate dropout supplements, with a yeast colony expressing the pair of proteins being analyzed. 将要分析的蛋白转化酵母组合克隆接种到2-5ml的SD溶液中 It is advisable to set up triplicate cultures for each type of yeast colony being analyzed. 3个重复 Fresh (one- to three-week-old) colonies will give best results for liquid culture inoculation. 最好是生长1-3周的克隆。 A single colony may be used for the inoculation if it is 2–3 mm in diameter. Scrape the entire colony into the medium. If the colonies on the master plate are smaller than 2 mm, transfer several colonies into the medium. 单一克隆最好是直径2-3mm,接种时需将它刮彻底，如果克隆小于2mm,可以刮多个克隆。 Examples: 比如： ? For AH109 and Y190 cotransformants expressing interacting pairs of GAL4 BD and AD fusion proteins,inoculate into SD/–His/–Leu/–Trp. AH109 和 Y190 菌株BD、AD共转生成融合蛋白可在SD/–His/–Leu/–Trp生长 ? For AH109 and Y190 cotransformants expressing non-interacting pairs of fusion protein constructs, inoculate into SD/–Leu/–Trp. AH109 和 Y190 菌株BD、AD共转不形成融合蛋白可在SD/–Leu/–Trp生长 ? For Y187 tranformants expressing interacting or non-interacting proteins, inoculate into SD/–Leu/–Trp. Y187 菌株的共转互作不互作都能在SD/–Leu/–Trp生长。 ? For non-transformed AH109, Y187, and Y190 host strains, inoculate into SD/–Ura. 三种菌株单转都可在SD/–Ura生长。 2. Incubate at 30°C overnight (~16–18 hr) with shaking (250 rpm). 30°C (~16–18 hr) 250 rpm 摇过夜。 3. Vortex the cell culture tube for 0