实验七. 聚丙烯酰胺凝胶等电聚焦电泳法【实验目的】1.掌握聚丙烯酰胺凝胶等电聚焦的基本原理2.学习用等电聚焦电泳测定蛋白质等电点的操作和方法【实验原理】等电聚焦法是一种特殊的聚丙烯酰胺凝胶电泳法。
它的特点是在凝胶柱中加入两性电解质载体-Ampholine，从而使凝胶柱上产生pH梯度。
当向两性载体凝胶施加电场时，即可形成pH梯度，pH梯度的顺序是从阳极到阴极pH值逐渐增大。
蛋白质为两性电解质，其所带电荷的性质和数量随所处环境的pH而变化。
当蛋白质在等电聚焦凝胶柱中进行电泳时，带电荷的蛋白质离子即在凝胶柱上泳动：带负电荷的蛋白质分子向阳极移动，带正电荷的蛋白质分子向阴极移动。
当蛋白质样品泳动到凝胶的某一部位，这一部位的pH值正好相当于该蛋白质的等电点时，蛋白质的净电荷为零不再移动，则聚焦形成一条蛋白质区带。
这种按等电点的大小在pH梯度某一相应位置进行聚焦的方法称为等电聚焦。
利用这种方法，在蛋白质聚焦的相应位置测定凝胶的pH值，就可得知该蛋白质的等电点。
【实验材料】1.实验器材小玻璃管：内径0.5cm，长10cm 2支；小玻管架；圆盘电泳槽；注射器和长针头；移液管；pH计2.实验试剂(1) 两性电解质载体凝胶：丙烯酰胺3.5g，N-甲叉双丙烯酰胺0.1g，pH3一10的Ampholine2.5ml，核黄素溶液（4mg/100ml）12.5ml，加水至50ml。
(2) 蛋白质溶液：纯牛血清白蛋白7mg，溶于1ml蒸馏水。
此蛋白溶液应无盐离子。
(3) 5％磷酸溶液(4) 2％氢氧化钠溶液(5) 考马斯亮蓝R-250染色液：称取考马斯亮蓝R-2500.25g，加入50%甲醇91ml和冰醋酸9ml。
(6) 40%蔗糖溶液(7) 12％三氯醋酸溶液(8) 脱色液：乙醇:冰醋酸:蒸馏水= 25：10：65(v／v)。
【实验操作】1. 取4ml两性电解质载体凝胶，置于抽气瓶中，加入0.07ml蛋白质溶液，轻轻摇动混匀，抽去气泡。
2. 取干净的小玻璃管2支，垂直放置，底端塞以橡皮塞，加入40％蔗糖溶液3—4滴，然后吸取抽气瓶中的两性电解质载体-蛋白质混合液1.8ml缓缓放入玻璃管中，加入胶液后立即用注射器加上一薄层水（3-5mm高），使混合液表面与空气隔绝。
放置约0.5-1小时，观察凝胶的凝聚情况。
3. 管内凝胶凝聚后，用滤纸将顶端水吸去，小心拔去管底橡皮塞，让蔗糖溶液流出，并用少量蒸馏水清洗，然后将小玻璃管垂直放入圆盘电泳槽中。
上槽加入5％磷酸溶液，接正极，下槽加入2% NaOH溶液，接负极，于150V条件下进行聚焦，过一段时间后（约2-3小时），电流稳定不变时（基本为零），说明聚焦完毕。
4. 聚焦结束后取出小玻璃管，迅速用蒸馏水将两端洗净。
用一带长针头的注射器灌满蒸馏水，并将针头紧贴玻璃管内壁插至凝胶和管壁之间，转动玻璃管，同时推动注射器，并使针头在管壁与凝胶间前进，注入蒸馏水，使凝胶胶条从玻璃管中脱出。
5. 将其中一根凝胶胶条浸于12%三氯醋酸溶液中固定2小时，白色蛋白区带即出现。
再浸于考马斯亮蓝染色液中染色5小时。
取出后，转移至脱色液中脱去背景颜色。
6. 将另一根凝胶胶条按顺序切成0.5cm长的小段，分别浸泡于1.0ml蒸馏水中过夜，用pH计测定pH值。
【实验结果】以凝胶胶条长度为横座标，pH值为纵座标，作图。
量出染色的各蛋白区带的距离，对照曲线查出其等电点。
【思考题】1.聚丙烯酰胺凝胶等电聚焦电泳法测定蛋白质等电点的原理？2. 聚丙烯酰胺凝胶等电聚焦电泳法操作时应注意的问题？Experiment 7. Polyacrylamide Gel Isoelectro Focusing【Purpose】1. Master the basic principle and operation of isoelectric focusing (IEF).2. Learn how to determine the isoelectric point of protein with IEF【Principle】Isoeletric focusing is a special kind of polyacylamide gel electrophoresis. Its character lies in the support of amphotericelectrolyte Ampholine and its addition into the gel column so that there is pH grade of the gel column. When the amphoteric support solution is imposed in the electric field, the pH gradient can be formed. And in pH gradient the pH increases gradually from the anode to the cathode.Protein molecule is a kind of amphoteric electrolyte. Its ion character and amount can change with the surrounding pH. When it migrates in the direct current electric field, the protein molecule with negative charge will migrate towards the anode, and the protein molecule with positive charge will migrate towards the cathode. The migration will stop at the pH correspondded with the PI of a protein and at this point, the protein has no charge and forms a band. Thus isoelectric focusing is a kind of electrophoresis action that according to isoelectric point, focuses at the corresponded position in the pH gradient.Thus the pI of a protein can be known by measuring the pH of the region, where the protein is focused.【Materials】1. Apparatus2 glass tubes (0.5×10), Rack for glass tube, Disc electrophoretic chamber, Injection syringe and long needle, Pipettes, pH meter.2. Reagents(1)Amphoteric electrolyte vector gel: 3g poluacylamide, 0.1g bisacrylamide, 2.5mlAmpholine(pH3-10), 12.5ml Hepatoflavin solution(4mg/100ml), add water to 50ml(2)Protein solution: 7mg pure bovine serum albumin, dissolved in 1ml distilled water. Thereshould be no ion in this solution.(3)5%phosphate solution(4)2% NaOH solution: 10g NaOH dissolved in little water, then dilute to 500ml(5)Commasie bright blue R250 solution: weigh 0.25g commasie bright blue, add 91ml 50%methanol and 9ml acetate.(6)40% sugar solution: weigh 4g sugar, dissolved in little water, then dilute to 10ml(7)12% trichloroacetic acid solution: weigh 12g trichloroacetic acid, dissolved in 100mldistilled water.(8)Decoloring solution: ethanol: acetate: distilled water = 25:10:65 (v/v) 【Procedures】1. Take 4ml Amphoteric electrolyte vector gel, add 0.07ml protein solution, shake gently to mix them and remove bubble with vacuum pump.2. Take 2 clean small glass tubes, place them vertically, and add rubber plugs on the bottoms.Add 3 to 4 drops of 40% sugar solution, then load 1.8ml Amphoteric electrolyte vector gel-protein mixture slowly into the glass tube. Immediately add a sheet of water on the gel with injection syringe to isolate the air from the mixture liquid after adding the gel solution. Then place for 30 to 60 minutes. The interface occurs first, then disappears, but occurs again, which indicates that the gel has been aggregated.3. After the gel has been fully aggregated, absorb the surface water with filtration paper. Draw out the rubber plug to let the sugar solution flow out, then wash it with little distilled water, and then place small glass tube in the disc electrophoretic chamber. Add 5% phosphate buffer to the upper chamber and this part is connected to the anode of power. Add 2% NaOH solution to the bottom chamber, and this part is connected to the cathode. Regulate the voltage to 150V. After 2 to3 hours, the current does not change (almost zero), which indicates the focusing is over.4. After focusing, take out the small glass tube and wash with distilled water immediately. Inject distilled water by injection syringe with long needle, and plug the needle in the inner wall of glass tube and the outer surface of gel, turn the glass tube around while pushing the syringe to make the needle forward between the inner surface of glass tube and outer surface of gel. Inject distilled water to make the gel column being expelled out of the small glass tube.5. Soak one of those gel columns into 12% trichloroacetic acid. About 2 hours later white protein zone appears. Then soak in commasie bright blue for 5 hours. Take the gel out and wash it.6. Cut every 0.5 cm of another gel column with sequence, soak in 1.0ml distilled water overnight, then measure the pH value with pH meter.【Results】The gel column length is x-axis, and the pH value is y-axis. Draw the curve. Measure the distance of every two protein band, check out the pI from the curve.【Questions】1. What is the principle of Polyacrylamide Gel Isoelectro focusing to measure the pI of protein?2. What are the attentions of Polyacrylamide Gel Isoelectro focusing?。