實驗六 聚丙烯酰胺凝膠電泳

1、.莈薂肄莄蒀螇羀莃薂薀袆莃節(jié)螆袂莂蒄蚈膀莁薇襖肆莀蠆蚇羂荿荿袂袈蒈蒁蚅膇蕆薃袀肅蕆蚅蚃罿蒆蒅衿羅肂薇 實驗八 聚丙烯酰胺凝膠圓盤電泳【實驗目的】1.掌握盤狀聚丙烯酰胺凝膠電泳的基本原理。2.學習盤狀聚丙烯酰胺凝膠電泳的操作技術，用于分離蛋白質。【實驗原理】CH2=CH聚丙烯酰胺凝膠電泳是以聚丙烯酰胺作為支持物的一種電泳形式。單體-丙烯酰胺和交聯劑-甲叉雙丙烯酰胺相互作用可形成聚丙烯酰胺，該聚合反應以TEMED作為催化劑，以APS 作為引發(fā)劑。丙烯酰胺和甲叉雙丙烯酰胺的比例可決定凝膠網孔的大小，交聯劑所占比重越大，凝膠的網孔就越小。 C=ONH2C=ONHCH2C=ONHCH2=CH+APTEM

2、EDNHCH2C=ONHCH2CHC=ONH2CH2CHC=ONH2CH2CHC=ONH2CH2CHC=ONH2丙烯酰胺甲叉雙丙烯酰胺聚丙烯酰胺 CH2=CH CH2 CHC=OCH2CH利用聚丙烯酰胺凝膠電泳進行蛋白質分離主要依據以下兩個因素： 蛋白質所帶靜電荷：在不同的pH條件下蛋白質所帶電荷不同。在一定的電場條件下蛋白質將向與其所帶電荷相反的電極方向移動，移動速率取決于蛋白質表面電荷的數量，電壓越強或電荷越多則蛋白質移動的越遠。其次，蛋白質的形狀和大?。旱鞍踪|在電泳中所受的阻力主要取決于樣品的大小與凝膠網孔大小之間的關系。蛋白質分子越小或凝膠網孔越大，所分離樣品所受阻力就愈小，則在電場中

3、的遷移率就越大。在非變性電泳中，天然蛋白質的分離就是蛋白質所帶電荷、分子大小及分子形狀等因素共同影響，作用的結果。電壓 x 樣品靜電荷遷移率 =  摩擦力濃縮效應可顯著提高聚丙烯酰胺凝膠電泳的分辨率，該效應可通過引入濃縮膠和不連續(xù)緩沖溶液系統而獲得。濃縮膠處于分離膠的頂部，因此樣品在進入分離膠之前首先要經過濃縮膠。它是由較低濃度的丙烯酰胺構成，當樣品經過濃縮膠時由于膠內網孔較分離膠網孔大，樣品的移動速度較快，最終使樣品“堆積”在濃縮膠和分離膠之間。 另外，濃縮膠還具有比分離膠更低的pH。濃縮膠內的緩沖溶液是Tris-HCl（pH 6.7）,該pH遠低于Tris的pK值(8.1)。分離

4、膠內的緩沖溶液是pH 8.9的Tris-HCl，而電泳正負兩極的緩沖溶液均為pH 8.3的Tris-甘氨酸緩沖溶液。在濃縮膠中，小分子并帶有大量負電荷的Cl-在凝膠中的移動速率較快，而電荷較少且分子量較大的甘氨酸的移動速率較慢。由于二者在同一電場中，具有相同的電流，由此形成的電壓梯度導致甘氨酸離子始終跟隨在Cl-的后面，帶有負電荷的蛋白質樣品則濃縮在甘氨酸離子和Cl-之間向正極移動。當樣品進入pH 8.9的分離膠時，甘氨酸的解離度增加，在電場中的遷移率高于樣品，蛋白質不再夾于兩種離子之間向正極移動，這時的蛋白質依靠分子篩效應和電荷效應進行分離。 樣品緩沖溶液 Tris/HCl pH 6.7Tr

5、is/甘氨酸 pH 8.9-+Tris/甘氨酸 pH 8.9分離膠 Tris/HCl pH 8.9濃縮膠 Tris/HCl pH 6.7【實驗材料】1. 實驗器材Bio-Rad公司Mini-Protean型電泳儀；電源（電壓200V，電流500mA）；100沸水浴；Eppendorf管；微量注射器（50l或100l）；帶蓋的玻璃或塑料小容器；搖床。2. 實驗試劑試劑號配方pH1分離膠緩沖溶液 1mol/L HCl 48.0 ml 三羥甲基氨基甲烷 36.3 g 加蒸餾水到100ml8.92單體交聯劑 丙烯酰胺 （Acr） 30g 甲叉丙烯酰胺（Bis） 0.8g 加蒸餾水到100ml3過硫酸銨

6、 100mg/ml4四甲基乙二胺（TEMED）5濃縮膠緩沖溶液 1mol/L HCl 48ml 三羥甲基氨基甲烷5.89 g 加蒸餾水到100ml6.76電極緩沖溶液 甘氨酸 28.8g三羥甲基氨基甲烷6.0 g 加蒸餾水到1000ml, 用前稀釋10倍8.37染色液 CBB G-250 0.1g 溶于95%乙醇后，加蒸餾水到100ml8示蹤染料 溴酚藍 0.05g溶于100ml 蒸餾水9樣品：蛇毒干粉200mg溶于20ml, pH6.7緩沖溶液（5）中，再加入25%蔗糖20ml和溴酚藍（8）10ml凝膠溶液的配方試劑分離膠（ml）濃縮膠(ml)125蒸餾水341.252.5-6.1950.0

7、50.005-1.01.257.640.100.005總體積（ml）1010Aa濃度（g%）7.53.0【實驗操作】 1.凝膠柱的制備 取l0cm×0.6cm的玻璃管，選擇較平整的一端為底端，量取7.5cm、8cm兩處，畫線；底端管口用小塊膠布封口，插入橡皮墊中，垂直放置于試管架中。用巴斯德滴管吸取分離膠，緩慢貼壁加膠到管內7.5cm處，立即加蒸餾水至8cm處。待分離膠凝固后，將膠面上的水分甩掉，殘留的水分用濾紙條吸干，用滴管速加濃縮膠到分離膠面上至8cm處，再小心地加一層覆蓋水。2.安裝電泳槽選擇無氣泡、無裂縫、長度合適的小玻璃管，撕掉膠布，安裝到電泳儀上，注意要緊密，以防止上槽緩

8、沖溶液漏液；向下槽注入電極緩沖液，注意用彎頭滴管除去玻璃管下端的氣泡；再向上槽倒入電極緩沖液淹沒小管，同樣用滴管除去氣泡。 3.加樣用微量注射器取樣品液30l，沿壁加在濃縮膠面上，注入時要慢，避免激起電極緩沖液。4.電泳連接電極，上槽與負極相連，下槽與正極相連；調節(jié)電流為lmA管，待示蹤染料進入分離膠時調節(jié)電流為2mA管，待示蹤染料接近凝膠管底部約0.5cm處，切斷電源，電泳時間為2小時3小時。5.剝膠取下凝膠管，用局麻針頭注射器吸取一定量的蒸餾水，將針頭插入膠柱-與管壁之間，邊注水邊旋轉玻管，直至膠柱與管壁分開，然后用洗耳球輕輕在玻管的一端加壓，使凝膠柱從玻管緩慢滑出，將凝膠柱置于編號的試管

9、內，用蒸餾水沖洗幾次。6.染色將考馬斯亮藍染色劑倒入放有膠條的小試管中，沒過膠條；60水浴保溫40分鐘50分鐘后取出，用水沖洗2次3次，觀察結果?！緦嶒灲Y果】觀察凝膠條中的蛋白質與考馬斯亮藍染色劑結合后所形成的藍色復合物，并通過畫圖記錄結果。【思考題】聚丙烯酰胺凝膠電泳分離生物大分子的基本原理？ 樣品液中加入蔗糖和溴酚藍的目的是什么？Experiment 8 Polyacrylamide Gel Electrophoresis in Cylindrical Tube【Purpose】 1 Master the principle polyacrylamide gel electrophores

10、is.2 Learn to use this approach of tube-polyacrylamide gel electrophoresis to separate the toxin of snake.【Principle】CH2=CHPolyacrylamide gel electrophoresis is a kind of electrophoresis in which the support media is polyacrylamide(PAGE). The organic monomer, acrylamide can react with cross-linking

11、reagent, methylene bisacrylamide to form polyacrylamide gel. This polymerizing reaction needs TEMED as catalyst and APS as arising agent. polyacrylamideCH2CH CH2 CHC=OCH2=CH C=ONH2NHCH2C=ONHCH2=CH+APTEMEDNHCH2C=ONHCH2CHC=ONH2CH2CHC=ONH2CH2CHC=ONH2CH2CHC=ONH2acarylamidemethylene bisacrylamideC=Opolya

12、crylamide 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 gel electrophoresis depends on two a

13、spects: 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 of their net surface charges. The h

14、igher 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. The smaller the size of the molecule

15、, 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 voltage x molecular chargemobility

16、 =  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 first through the stackin

17、g 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 highly porous and then "stack" up at the interface between the two gels since the

18、 running gel has much smaller pores. Stacking gel Tris/HCl pH 6.7Running gel Tris/HCl pH 8.9Tris/Glycine pH 8.9Sample in Tris/HCl pH 6.7+-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 Tris (8.1). The run

19、ning 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 throughout this electric

20、al 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 becomes more signific

21、antly 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-Rad Mini-Protean apparatus; Power supply (capacity 200V, 500MA); B

22、oiling water bath; Eppendorf centrifuge (optional); Hamilton Syringes (50l and 100l capacity); Small glass or plastic container with lid (i.e.12cm×16 cm×3cm); Rocking or rotary shaker.2. ReagentsregentmenupH1Separating gel buffer: 1mol/L HCl 48.0 ml, Tris 36.3 g, add distilled water to 100

23、ml.8.92Stock acrylamide solution: 30 g acrylamide, 0.8 g bis-acrylamide. Make up to 100 ml in distilled water.310% Ammonium persulfate in distilled water.4N,N,N',N'-tetramethylethylenediamine (TEMED).5Stacking gel buffer: 48ml 1mol/L HCl, Tris5.89 g add distilled water to 100ml.6.7610×E

24、lectrophoresis 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 H20 and 10ml glacial acetic acid.8Footprint dye bromophenol blue：bromophenol blue 0.05g in 100ml distilled w

25、ater.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）Stacking gel (ml)125Distilled water341.252.5-6.1950.050.005-1.01.257.640.100.005Total volumes （ml）1010The con

26、centration of the gel（g%）7.53.0【Procedures】1. Preparation of the gelClean the internal surfaces of the glass tube (10 cm×0.6cm) 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

27、(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 water on top of the separating gel until the position 8.0 cm immediately. When the polymerization

28、 has finished in 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 di

29、stilled 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 airtightly. Discard the bottom plaster, and move the bubble under the bottom. Add electrophoresis bu

30、ffer to inner and outer reservoir, making sure that both top and bottom of gel are immersed in buffer.3. Loading SamplesLoad 30l sample solution 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 towards the anode; Turn on power supply