G α-Gal Quantitative Assay

1、G. -Gal Quantitative AssayG. -Gal定量分析Reagents and Materials Required:材料與試劑： Appropriate liquid synthetic dropout (SD) culture medium (Appendix C.A)適量SD液體培養(yǎng)基 50-ml culture tubes 50ml 培養(yǎng)管 PNP-Gal Solution (100 mM) 100 mM PNP-Gal溶液 10X Stop Solution (Appendix D.F) 10X 終止液 （1 M Na2CO3溶液） 1X NaOAc Buffer

2、 (Appendix D.F) 0.5M 醋酸鈉 Assay Buffer (Appendix D.F) 分析緩沖液：現(xiàn)配 0.5M 醋酸鈉溶液與100 mM PNP-Gal溶液按體積比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 酶標(biāo)儀Preparation of Samples樣品準(zhǔn)備：1. Inoculate 25 ml of liquid synthetic dropout (SD) medium, containing

3、 the appropriate dropout supplements, with a yeast colony expressing the pair of proteins being analyzed.將要分析的蛋白轉(zhuǎn)化酵母組合克隆接種到2-5ml的SD溶液中It is advisable to set up triplicate cultures for each type of yeast colony being analyzed. 3個重復(fù)Fresh (one- to three-week-old) colonies will give best results for liq

4、uid culture inoculation. 最好是生長1-3周的克隆。A single colony may be used for the inoculation if it is 23 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.單一克隆最好是直徑2-3mm,接種時需將它刮徹底，如果克隆小于2mm,可以刮多個克隆。

5、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共轉(zhuǎn)生成融合蛋白可在SD/His/Leu/Trp生長 For AH109 and Y190 cotransformants expressing non-interacting pairs of fusion protein constructs, inoculate int

6、o SD/Leu/Trp.AH109 和 Y190 菌株BD、AD共轉(zhuǎn)不形成融合蛋白可在SD/Leu/Trp生長 For Y187 tranformants expressing interacting or non-interacting proteins, inoculate into SD/Leu/Trp. Y187 菌株的共轉(zhuǎn)互作不互作都能在SD/Leu/Trp生長。 For non-transformed AH109, Y187, and Y190 host strains, inoculate into SD/Ura. 三種菌株單轉(zhuǎn)都可在SD/Ura生長。2. Incubate a

7、t 30°C overnight (1618 hr) with shaking (250 rpm). 30°C (1618 hr) 250 rpm 搖過夜。3. Vortex the cell culture tube for 0.51 min to disperse cell clumps, then transfer 1 ml of the suspension to a clean cuvette and record the OD600. For accuracy, the OD600 should lie between 0.51.0. Dilute the ce

8、ll suspension if necessary; remember to account for the dilution factor when making your final calculations (Step 11, below). 渦旋震蕩0.5-1min培養(yǎng)液,然后測量其OD600，其值應(yīng)在0.5-1.0之間，若過濃則稀釋。下面11步同理。4. Place 1.0 ml of culture into a 1.5-ml microcentrifuge tube. Centrifuge at 14,000 rpm (10,000 x g) for 2 min or unti

9、l the cells are completely pelleted. 取1ml溶液只1.5ml離心管14000rpm離心2min至細(xì)胞完全沉淀。Note: It is important to ensure that cells and cellular debris are completely pelleted to minimize intereference from light scattering in the colorimetric assay below.注意：細(xì)胞和細(xì)胞碎片充分離下來對下面比色分析很重要，確保離心充分。5. Carefully transfer the

10、supernatant to a clean tube and store at room temperature for use in Step 6, below.小心轉(zhuǎn)移上清至新離心管室溫保存，接著做第6步。Note: To minimize the loss of enzyme activity, we suggest proceeding with the colorimetric assay immediately once the cell-free superantant has been isolated.注意：為了限制酶活性，我們建議取上清后馬上進(jìn)行比色分析。Colorime

11、tric Assay比色分析：Below we provide protocols for 1-ml and 200-l assays. If you have access to a spectrophotometer equipped to read microtiter plates, you may find it more convenient to use the 200-l assay protocol, which is intended for use with 96-well, flat-bottom microtiter plates.以下提供1ml及200l 2種分析方

12、法，如果有合適的比色皿或儀器，更提倡使用200l的方法，甚至可以使用96孔板來。Note: The experimental conditions and volumes of reagents used throughout the -Galactosidase Quanitative Assay have been carefully tested and optimized for use in the 1-ml and 200-l assay formats described below. Please follow as directed. Though we do not rec

13、ommend changing the actual volumes of reagents used in the colorimetric assay, if the signal from an experimental sample exceeds the linear range of the assay, you can dilute the media supernatant before transferring an aliquot to the reaction tube or well. Remember to correct for individual sample

14、volumes and dilutions when tabulating final results at Step 11.從頭到尾分析及溶液體積使用統(tǒng)一標(biāo)準(zhǔn)。5. Prepare a sufficient amount of Assay Buffer for all samples including controls, and allow to equilibrate to room temperature.充分準(zhǔn)備緩沖液穩(wěn)定室溫保存。 For each 1-ml assay, you will need 24 l Assay Buffer.采用1ml樣品分析量則需要24l緩沖液/ml。

15、 For each 200-l assay (96-well microtiter plate format), you will need 48 l Assay Buffer.若采用200l方法分析，如96孔板分析，每個樣則需要48l緩沖液。Note: We recommend assaying each sample in triplicate. Be sure to include positive and negative controls in your assay. Also include a reagent blank in each assay to calibrate th

16、e spectrophotometer prior to reading the OD of your samples. A reagent blank is composed of sterile, unused culture media, Assay Buffer, and Stop Solution combined according to Steps 69.推薦使用3個重復(fù)及需加陰陽對照，用相應(yīng)溶液調(diào)OD值，根據(jù)6-9步，準(zhǔn)備相應(yīng)的空白溶液，緩沖液，終止液等。Assay Scale 200-l 分析量分別為 200l 6. Transfer cell culture medium

17、supernatant (from Step 4) 16 l into a well of a clear microtiter platea. 轉(zhuǎn)移上清（接第4步）16 l干凈的96孔板7. Add Assay Buffer to each sample. 48 l每個樣加48l緩沖液8. Incubate at 30°C for 60 min. Be sure to cover microtiter plates with a lid or parafilm to prevent evaporation. 30°C 溫育60min,蓋蓋或封板防蒸發(fā)。9. Termina

18、te the reaction by adding Stop Solution. 136 l of 10X 加136 l 10X終止液終止反應(yīng)。Note: Use 10X Stop Solution for 200-l assays.200 l 分析用10X10. Record the optical density of each sample at 96-well plate 410 nm (OD410)測OD410。a Add a corresponding volume of sterile, unused culture media to the reagent blank.a.用未

19、接菌培養(yǎng)液作為空白對照b Zero the spectrophotometer using the reagent blank and measure the OD410 of the experimental and control samples relative to the blank.b.空白對照的測量值作為0點。11. Calculate -galactosidase units. One unit of -galactosidase is defined as the amount of enzyme that hydrolyzes 1 mole p-nitrophenyl-d-

20、galactoside to p-nitrophenol and d-galactose in 1 min at 30°C in acetate buffer, pH 4.5 (Lazo et al., 1978).-gal的計算單位。在，30°C， pH 4.5的醋酸鹽緩沖液中, 1 molPNP-Gal 在-gal作用下1 min分解為PNP Gal及D-gal被定義為1單位的-gal 。-galactosidase milliunits/(ml x cell) = OD410 x Vf x 1,000/( x b) x t x Vi x OD600t = elapse

21、d time (in min) of incubation 孵化時間（分鐘計）Vf= final volume of assay (200 l )終體積（200l）Vi= volume of culture medium supernatant added (16 l )加入的樣液體積（16l）OD600 = optical density of overnight culturea OD600值 x b = p-nitrophenol molar absorbtivityb at 410 nm x the light path (cm)= 10.5 (ml/mol) for 200-l fo

22、rmat b,c x b = 10.5 (ml/mol) a The optical density at 600 nm, recorded in Step 2, is used to normalize the OD410 of different media samples to the number of cells in each culture.a. 第2步溶液測量出的OD600的作為OD410的標(biāo)準(zhǔn)值。b. The molar absorbtivity, though independent of concentration and light path, varies with

23、the chemical properties (e.g., pH) of the solution. Because different strengths and quantities of Stop Solution are used to terminate the 200-and 1-ml reactions, the final pHs and, therefore, the molar absorbtivities are different for the two formats.c. Determined at Clontech using a SPECTRAmaxR Mic

24、roplate Spectrophotometer and Corning Costar UV-transparent, flat-bottom plates (Corning Cat No. 3635); Some microplate readers have pathlength correction capabilities to normalize absorbance values to those obtained when the light pathlength is 1-cm (e.g., using 1.5-ml cuvettes). With pathlength correction on, the molar absorbtivity, , of p-nitrophenol at 410 nm in the 200-l format was determined to be 20.3 ml/mol.The well-diameter and, therefore,