实验八聚丙烯酰胺凝胶圆盘电泳.docx

1、实验八聚丙烯酰胺凝胶圆盘电泳实验八 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】CH2=CH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=CH CH2 CHCH2CH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

2、所带电荷不同。在一定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

3、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

4、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。样品缓冲溶液 Tris/HCl pH 6.7Tris/甘氨酸 pH 8.9-+Tris/甘氨酸 pH 8.9分离胶 Tris/HCl pH 8.9浓缩胶 Tris/HCl pH 6.7【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

5、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pH1分离胶缓冲溶液 1mol/L HCl 48.0 ml 三羟甲基氨基甲烷 36.3 g 加蒸馏水到100ml8.92单体交联剂 丙烯酰胺 （Acr） 30g 甲叉丙烯酰胺（Bis） 0.8g加蒸馏水到100ml3过硫酸铵 100mg/ml4四甲基乙二胺（TEMED）5浓缩胶缓冲溶液 1mol/L HCl 48ml 三羟甲基氨基甲烷5.89 g 加蒸馏水到100ml6.76电极缓冲溶液 甘氨酸 28.8g三羟甲基氨基甲烷6.0 g加蒸馏水到1000ml, 用前稀释10倍8.37染色液 CBB G-250 0.1g 溶于95%

6、乙醇后，加蒸馏水到100ml8示踪染料 溴酚蓝0.05g溶于100ml 蒸馏水9样品：蛇毒干粉200mg溶于20ml, pH6.7缓冲溶液（5）中，再加入25%蔗糖20ml和溴酚蓝（8）10ml凝胶溶液的配方试剂分离胶（ml）浓缩胶(ml)125蒸馏水341.252.5-6.1950.050.005-1.01.257.640.100.005总体积（ml）1010Aa浓度（g%）7.53.0【实验操作】1.凝胶柱的制备 取l0cm0.6cm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

7、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

8、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

9、是什么？Experiment 8 Polyacrylamide Gel Electrophoresis in Cylindrical Tube【Purpose】1 Master the principle polyacrylamide gel electrophoresis.2 Learn to use this approach of tube-polyacrylamide gel electrophoresis to separate the toxin of snake.【Principle】CH2=CHPolyacrylamide gel electrophoresis is a ki

10、nd of electrophoresis in which the support media is polyacrylamide(PAGE). The organicmonomer, 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. CH2CH CH2 CHCH2=CHThe propor

11、tion 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 gel electrophoresis depends on two aspects:Net charges on

12、 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 of their net surface charges.The higher the voltage or th

13、e 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. The smaller the size of the molecule, or the larger the size

14、 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 voltage x molecular chargemobility = molecular frictionThe

15、 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 first through the stacking gel. Stacking gel is made us

16、ing 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 highly porous and thenstack up at the interface between the two gels since the running gel has much smaller pores.Tris/Gl

17、ycine 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 Tris (8.1). The running gel is a Tris-HCl buffer at pH 8.9 and the running bufferfor the gel overall is Tris-glycine pH 8.3. In stacking gel, the fully-charge chlor

18、ide will move fast through theporous. The larger and slightly charged glycine will moveslowly. But the current must be the same throughout this electricalcircuit. A voltage gradient allows the glycine to remain just behindthe chloride ions. The proteins with negativechargewill migrate between the ch

19、loride and theglycine, in very sharp bands.When the samples enter the running gel with the pH increasing to 8.9, the glycine becomes more significantly deprotonated and it movesahead of the proteins.The proteins are now not forced to stack between the two ions, andcan proceed to be separated by the

20、molecular sieving of the higherconcentration gel.【Materials】1. ApparatusBio-Rad Mini-Protean apparatus; Power supply (capacity 200V, 500MA); Boiling water bath; Eppendorf centrifuge (optional); Hamilton Syringes (50l and 100l capacity); Small glass or plastic container with lid (i.e.12cm16cm3cm); Ro

21、cking or rotary shaker.2. ReagentsregentmenupH1Separating gel buffer: 1mol/L HCl 48.0 ml, Tris 36.3 g,add distilled water to 100ml.8.92Stock acrylamide solution:30 g acrylamide, 0.8 gbis-acrylamide. Make up to 100 ml in distilled water.310% Ammonium persulfate in distilled water.4N,N,N,N-tetramethyl

22、ethylenediamine (TEMED).5Stacking gel buffer: 48ml 1mol/L HCl, Tris5.89 g add distilled water to 100ml.6.7610Electrophoresis buffer: Dissolve 6.0 g of Tris base and 28.8 g of glycine in water and adjust the volume to 1 L. 8.37Coomassie Gel Stain:Add 0.1g Coomassie Blue R-250 into 45ml methanol, 45ml

23、 H20 and 10ml glacial acetic acid.8Footprint dye bromophenol blue：bromophenol blue 0.05g in 100ml distilled water.9Sample:toxin of snake 200mg dissolve in 20ml, pH6.7 stacking gel buffer,and add 25%sucrose 20ml and bromophenol blue 10ml.Preparation of Acrylamide Solutions regentseparating gel（ml）Sta

24、cking gel (ml)125Distilled water341.252.5-6.1950.050.005-1.01.257.640.100.005Total volumes （ml）1010The concentration of the gel（g%）7.53.0【Procedures】1.Preparation of the gelClean the internal surfaces of the glass tube(10cm0.6cm) and dry, then make markers at 7.5cm and 8.0cm.Envelop the bottom with

25、plastertightly,then put it into cushion, and clamp it in a vertical position. Using a Pasteur (or larger) pipet to transfer separating gel mixture to the tube by running the solution carefully down the edge. Continue to add this solution until it reaches the position 7.5 cm,gently lay distilled wate

26、r on top of the separating gel until the position 8.0 cm immediately. When the polymerization has finished in running gel, pour off the overlaying waterand 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 t

27、op of the stacking gel. The same point also should be noticed as last step which prevents distilled water from 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 airtight

28、ly.Discard the bottomplaster,and move the bubble under the bottom. Add electrophoresis buffer to inner and outer reservoir, making sure that both top and bottom of gel are immersed in buffer.3. Loading SamplesLoad 30l sample solutionwith minim syringe,carefully down the edge to the surface of stacki

29、ng gel.4. Running a GelAttach electrode plugs to proper electrodes, Anode should connect with inner reservior and current should flow towards the anode;Turn on power supply to 1 mA /tube when the dye migratesin stacking gel and change the current to 2mA when the dye migrates to separating gel. When

30、the dye migrates to0.5cm from the bottom of the gel, turn off power supply.5. Peel off the gelRemove cylindrical tubefrom electrode assembly firstly and use a syringe with 10cm long pinhead full of water as lube. Put the long pinhead into the spacer between gel and glass, injecting and turning until

31、 the gel is separated from the tube, and blow gently to make the gel slip out.6. Staining the Gel Put the gel into little tube with Coomassie brilliant blue, and incubate at 60 for 40min -50min, then take out and wash with water for two or three times .【Result】Observe the blue compound which formed between protein and Coomassie brilliant blue in the gel anddraw out the result. 【Questions】What is the separating principle of p