2020年聚丙烯酰胺圆盘精品版.doc

1、精选文档实验八 聚丙烯酰胺凝胶圆盘电泳【实验目的】1. 掌握盘状聚丙烯酰胺凝胶电泳的基本原理。2. 学习盘状聚丙烯酰胺凝胶电泳的操作技术，用于分离蛋白质。【实验原理】聚丙烯酰胺凝胶电泳是以聚丙烯酰胺作为支持物的一种电泳形式。单体 -丙烯酰胺和交联剂-甲叉双丙烯酰胺相互作用可形成聚丙烯酰胺，该聚合反应以 TEMED （四甲基乙胺）作为催化剂，以 APS 作为引发剂。丙烯酰胺和甲叉双丙烯酰胺的比例可决定凝胶网孔的大小，交联剂所占比重越大，凝胶的网孔就越小。CH2=CC=OCH2=CNHC=O+APCH2NH2TEMEDNH丙烯酰胺C=OCH2=C甲叉双丙烯酰胺CH2CH CH2CH CH2 CHC

2、=OC=OC=ONH2NH2NHCH2NHC=OCH2CCH2CH CH2CHC=OC=ONH2NH2聚丙烯酰胺利用聚丙烯酰胺凝胶电泳进行蛋白质分离主要依据以下两个因素：蛋白质所带静电荷：在不同的pH 条件下蛋白质所带电荷不同。在一定精选文档精选文档的电场条件下蛋白质将向与其所带电荷相反的电极方向移动，移动速率取决于蛋白质表面电荷的数量，电压越强或电荷越多则蛋白质移动的越远。其次，蛋白质的形状和大小：蛋白质在电泳中所受的阻力主要取决于样品的大小与凝胶网孔大小之间的关系。蛋白质分子越小或凝胶网孔越大，所分离样品所受阻力就愈小，则在电场中的迁移率就越大。在非变性电泳中，天然蛋白质的分离就是蛋白质所

3、带电荷、分子大小及分子形状等因素共同影响，作用的结果。电压x 样品静电荷迁移率=摩擦力浓缩效应可显著提高聚丙烯酰胺凝胶电泳的分辨率，该效应可通过引入浓缩胶和不连续缓冲溶液系统而获得。浓缩胶处于分离胶的顶部，因此样品在进入分离胶之前首先要经过浓缩胶。它是由较低浓度的丙烯酰胺构成，当样品经过浓缩胶时由于胶内网孔较分离胶网孔大，样品的移动速度较快，最终使样品“堆积”在浓缩胶和分离胶之间。另外，浓缩胶还具有比分离胶更低的pH 。浓缩胶内的缓冲溶液是Tris-HCl （ pH 6.7 ）,该 pH远低于 Tris的 pK 值 (8.1)。分离胶内的缓冲溶液是 pH 8.9的 Tris-HCl ，而电泳正

4、负两极的缓冲溶液均为 pH 8.3的 Tris- 甘氨酸缓冲溶液。在浓缩胶中，小分子并带有大量负电荷的 Cl -在凝胶中的移动速率较快，而电荷较少且分子量较大的甘氨酸的移动速率较慢。由于二者在同一电场中，具有相同的电流，由此形成的电压梯度导致甘氨酸离子始终跟随在Cl -的后面，带有负电荷的蛋白质样品则浓缩在甘氨酸离子和Cl -之间向正极移动。当样品进入 pH 8.9 的分离胶时，甘氨酸的解离度增加，在电场中的迁移率高于样品，蛋白质不再夹于两种离子之间向正极移动，这时的蛋白质依靠分子筛效应和电荷效应进行分离。精选文档精选文档Tris/ 甘氨酸 pH 8.9-样品缓冲溶液浓缩胶Tris/HCl p

5、H 6.7Tris/HCl pH 6.7分离胶Tris/HCl pH 8.9Tris/ 甘氨酸pH 8.9+【实验材料】1. 实验器材Bio-Rad 公司 Mini-Protean 型电泳仪； 电源（电压 200V ，电流 500mA ）；100沸水浴； Eppendorf 管；微量注射器（ 50l或 100l）；带盖的玻璃或塑料小容器；摇床。2. 实验试剂试剂号配方pH分离胶缓冲溶液1mol/L HCl48.0 ml1三羟甲基氨基甲烷36.3 g8.9加蒸馏水到 100ml单体交联剂丙烯酰胺 （ Acr ）30g2甲叉丙烯酰胺（ Bis ） 0.8g加蒸馏水到 100ml3过硫酸铵100mg

6、/ml4 四甲基乙二胺（ TEMED ）浓缩胶缓冲溶液1mol/L HCl48ml5三羟甲基氨基甲烷5.89 g6.7加蒸馏水到 100ml电极缓冲溶液甘氨酸28.8g6三羟甲基氨基甲烷6.0 g8.3加蒸馏水到 1000ml,用前稀释10 倍染色液CBB G-250 0.1g溶于 95%乙醇后，加7蒸馏水到 100ml精选文档精选文档8示踪染料溴酚蓝 0.05g 溶于 100ml蒸馏水样品：蛇毒干粉200mg 溶于 20ml, pH6.7 缓冲溶液（5）925%蔗糖 20ml 和溴酚蓝（ 8）10ml中，再加入凝胶溶液的配方试剂分离胶（ ml ）浓缩胶 (ml)11.25-22.51.05-

7、1.25蒸馏水6.1957.6430.050.1040.0050.005总体积（ ml）1010Aa 浓度（ g% ）7.53.0【实验操作】1. 凝胶柱的制备取 l0cm 0.6cm 的玻璃管，选择较平整的一端为底端，量取7.5cm、8cm 两处，画线；底端管口用小块胶布封口，插入橡皮垫中，垂直放置于试管架中。用巴斯德滴管吸取分离胶，缓慢贴壁加胶到管内7.5cm 处，立即加蒸馏水至 8cm 处。待分离胶凝固后，将胶面上的水分甩掉，残留的水分用滤纸条吸干，用滴管速加浓缩胶到分离胶面上至8cm 处，再小心地加一层覆盖水。2. 安装电泳槽选择无气泡、无裂缝、长度合适的小玻璃管，撕掉胶布，安装到电泳

8、仪上，注意要紧密，以防止上槽缓冲溶液漏液；向下槽注入电极缓冲液，注意用弯头滴管除去玻璃管下端的气泡；再向上槽倒入电极缓冲液淹没小管，同样用滴管除去气泡。3. 加样用微量注射器取样品液 30l ，沿壁加在浓缩胶面上，注入时要慢，避免激起电极缓冲液。4. 电泳连接电极，上槽与负极相连，下槽与正极相连；调节电流为lmA 管，待示踪染料进入分离胶时调节电流为2mA 管，待示踪染料接近凝胶管底部约 0.5cm 处，切断电源，电泳时间为2 小时 3 小时。5. 剥胶取下凝胶管， 用局麻针头注射器吸取一定量的蒸馏水，将针头插入胶柱-与管壁之间，边注水边旋转玻管，直至胶柱与管壁分开，然后用洗耳球轻精选文档精选

9、文档轻在玻管的一端加压，使凝胶柱从玻管缓慢滑出，将凝胶柱置于编号的试管内，用蒸馏水冲洗几次。6. 染色将考马斯亮蓝染色剂倒入放有胶条的小试管中，没过胶条；60水浴保温 40 分钟 50 分钟后取出，用水冲洗2 次 3 次，观察结果。【实验结果】观察凝胶条中的蛋白质与考马斯亮蓝染色剂结合后所形成的蓝色复合物，并通过画图记录结果。【思考题】聚丙烯酰胺凝胶电泳分离生物大分子的基本原理？样品液中加入蔗糖和溴酚蓝的目的是什么？精选文档精选文档Experiment 8Polyacrylamide Gel Electrophoresis inCylindrical Tube【Purpose】 Master

10、the principle polyacrylamide gel electrophoresis. Learn to use this approach of tube-polyacrylamide gel electrophoresis to separate the toxin of snake.【 Principle】Polyacrylamide gel electrophoresis is a kind of electrophoresis in which the support media is polyacrylamide(PAGE). The organic monomer,

11、acrylamide can react with cross-linking reagent, methylene bisacrylamide to form polyacrylamide gel. This polymerizing reaction needs TEMED as catalyst and APS as arising agent.CH2=CC=OCH2=CNHC=O+APCH2NH 2TEMEDNHacarylamideC=OCH2=CHmethylene bisacrylamideCH2CH CH2CH CH2 CHC=OC=OC=ONH2NH2NHCH2NHC=OCH

12、2CCH2CHCH2CHC=OC=ONH2NH2精选文档polyacrylamide精选文档The proportion between acrylamide and methylene bisacrylamide results in the size of pores in the gel, smaller pores sizes are obtained by using a higher concentration of cross-linking reagent to form the gel.The separation of protein in polyacrylamide g

13、el electrophoresis depends on two aspects:Net charges on the proteins: Depending on the pH of the buffer, proteins in a sample will carry different charges. When an electric field is applied, proteins will migrate towards their corresponding poles. The rate of migration will depend on the strength o

14、f their net surface charges. The higher the voltage or the greater the charge on the protein the further it will moveShape and size of the Proteins: The friction of a protein is largely determined by the relationship between the effective size of the molecule and the size of the pores in the gel. Th

15、e smaller the size of the molecule, or the larger the size of the pores in the gel, the lower the resistance and therefore the faster a molecule moves through the gel.In non-denaturing electrophoresis, the native proteins are separated based on a combination of their charge, size and shape.applied v

16、oltage x molecular chargemobility =molecular frictionThe resolution of separation in electrophoresis can be improved by the use of a stacking gel and a discontinuous buffer system, which contributes to the stacking effect.The stacking gel resides on top of the running gel, and thus the sample passes

17、 first through the stacking gel. Stacking gel is made using a lower percentage of acrylamide than the running gel and it has less molecular sieving. So, after loading samples, the proteins run rapidly through the stacking gel which is精选文档精选文档highly porous and then stack up at the interface between t

18、he two gels since the running gel has much smaller pores.Tris/Glycine pH 8.9-Sample inStacking gelTris/HCl pH 6.7Tris/HCl pH 6.7Running gelTris/HCl pH 8.9+Tris/Glycine pH 8.9The stacking gel also has a lower pH than the running gel. It is a Tris-HCl buffer at pH 6.7, which is much below the pK of Tr

19、is (8.1). The running gel is a Tris-HCl buffer at pH 8.9 and the running buffer for the gel overall is Tris-glycine pH 8.3. In stacking gel, the fully-charge chloride will move fast through the porous. The larger and slightly charged glycine will move slowly. But the current must be the same through

20、out this electrical circuit. A voltage gradient allows the glycine to remain just behind the chloride ions. The proteins with negative charge will migrate between the chloride and the glycine, in very sharp bands. When the samples enter the running gel with the pH increasing to 8.9, the glycine beco

21、mes more significantly deprotonated and it moves ahead of the proteins. The proteins are now not forced to stack between the two ions, and can proceed to be separated by the molecular sieving of the higher concentration gel.【 Materials 】1. ApparatusBio-RadMini-Proteanapparatus; Power supply (capacit

22、y 200V, 500MA);Boiling water bath; Eppendorf centrifuge (optional); Hamilton Syringes (50 l and 100 lcapacity) ; Small glass or plastic container with lid (i.e.12cm 16 cm3cm); Rocking or rotary shaker.精选文档精选文档2. Reagentsregent12menuSeparating gel buffer: 1mol/L HCl48.0 ml, Tris 36.3 g, adddistilled

23、water to 100ml.Stockacrylamidesolution:30gacrylamide,0.8gpH8.9bis-acrylamide. Make up to 100 ml in distilled water.3 10% Ammonium persulfate in distilled water.4 N,N,N,N -tetramethylethylenediamine (TEMED).5678Stackinggel buffer:48ml1mol/LHCl,Tris5.89g adddistilled water to 100ml.10Electrophoresis b

24、uffer: Dissolve 6.0 g of Tris base and28.8 g of glycine in water and adjust the volume to 1 L.Coomassie Gel Stain: Add 0.1g Coomassie Blue R-250 into45ml methanol, 45ml H 20 and 10ml glacial acetic acid.Footprint dye bromophenol blue ：bromophenol blue 0.05g in100ml distilled water.Sample: toxinof sn

25、ake 200mg dissolvein 20ml,pH6.76.78.39 stacking gel buffer, and add 25% sucrose 20ml and bromophenol blue 10ml.Preparation of Acrylamide Solutionsregentseparating gel（ml ）Stacking gel (ml)11.25-22.51.05-1.25Distilled water6.1957.6430.050.1040.0050.005Total volumes （ml ）1010The concentration of7.53.0

26、the gel（ g%）【 Procedures】1. Preparation of the gelClean the internal surfaces of the glass tube (10 cm0.6cm) and dry, then make markers at 7.5cm and 8.0cm. Envelop the bottom with plaster tightly, then put it into cushion, and clamp it in a vertical position. Using a Pasteur (or larger) pipet to tra

27、nsfer separating gel mixture to the tube by running the solution精选文档精选文档carefully down the edge. Continue to add this solution until it reaches the position 7.5 cm, gently lay distilled water on top of the separating gel until the position 8.0 cm immediately. When the polymerization has finished in

28、running gel, pour off the overlaying water and then add the stacking gel solution to the tube until the solution reaches to the position 8.0 cm. gently lay about 0.5cm of distilled water on top of the stacking gel. The same point also should be noticed as last step which prevents distilled water fro

29、m mixing with gel solution. Allow stacking gel to polymerize (about 30 minutes).2. The installation of the gelPlace the eligible gel tube into electrophoresis chamber, vertically and airtightly. Discard the bottom plaster, and move the bubble under the bottom. Add electrophoresis buffer to inner and

30、 outer reservoir, making sure that both top and bottom of gel are immersed in buffer.3. Loading SamplesLoad 30 l samplesolution with minim syringe, carefully down the edge to the surface of stacking gel.4. Running a GelAttach electrode plugs to proper electrodes, Anode should connect with inner reservior and current should flow