生物化学实验讲义（考研资料）：实验四 电泳

实验四 蛋白质的聚丙烯酰胺凝胶电泳 最广泛使用的不连续缓冲系统最早是由Ornstein(1964) 和Davis(1964) 设计的, 样品和浓缩胶中含 Tris-HCl(pH 6.8), 上下槽缓冲液含Tris-甘氨酸(pH 8.3), 分离胶中含Tris-HCl(pH 8.8)。系统中所有组分都含有0.1% 的 SDS(Laemmli, 1970)。样品和浓缩胶中的氯离子形成移动界面的先导边界而甘氨酸分子则组成尾随边界，在移动界面的两边界之间是一电导较低而电位滴度较陡的区域, 它推动样品中的蛋白质前移并在分离胶前沿积聚。此处pH值较高， 有利于甘氨酸的离子化，所形成的甘氨酸离子穿过堆集的蛋白质并紧随氯离子之后，沿分离胶泳动。从移动界面中解脱后，SDS-蛋白质复合物成一电位和pH值均匀的区带泳动穿过分离胶，并被筛分而依各自的大小得到分离。 SDS与蛋白质结合后引起蛋白质构象的改变。SDS-蛋白质复合物的流体力学和光学性质表明，它们在水溶液中的形状，近似于雪茄烟形状的长椭园棒，不同蛋白质的SDS复合物的短轴长度都一样（约为18?，即1.8nm），而长轴则随蛋白质分子量成正比地变化。这样的SDS-蛋白质复合物，在凝胶电泳中的迁移率，不再受蛋白质原有电荷和形状的影响，而只是椭园棒的长度也就是蛋白质分子量的函数。 由于SDS和巯基乙醇的作用，蛋白质完全变性和解聚，解离成亚基或单个肽链，因此测定的结果只是亚基或单条肽链的分子量。 SDS聚丙烯酰胺凝胶的有效分离笵围取决于用于灌胶的聚丙烯酰胺的浓度和交联度。在没有交联剂的情况下聚合的丙烯酰胺形成毫无价值的粘稠溶液，而经双丙烯酰胺交联后凝胶的刚性和抗张强度都有所增加，并形成SDS蛋白质复合物必须通过的小孔。这些小孔的孔径随 “双丙烯酰胺～丙烯酰胺” 比率的增加而变小，比率接近 1：20 时孔径达到最小值。SDS聚丙烯酰胺凝胶大多按“双丙烯酰胺～丙烯酰胺”为1：29 配制，试验表明它能分离大小相差只有3% 的蛋白质。 凝胶的筛分特性取决于它的孔径，而孔径又是灌胶时所用丙烯酰胺和双丙烯酰胺绝对浓度的函数。用5～15%的丙烯酰胺所灌制凝胶的线性分离范围如下表： SDS聚丙烯酰胺凝胶的有效分离范围 *丙烯酰胺浓度（%） 线性分离范围（kD）  15 12～43  10 16～68  7.5 36～94  5.0 57～212  *双丙烯酰胺～丙烯酰胺摩尔比为 1：29。 1. SDS聚丙烯酰胺凝胶的配制 ⑴ 试剂 ①丙烯酰胺和N, N’-亚甲双丙烯酰胺。以温热（利于溶解双丙烯酰胺）的去离子水配制含有29%（w/v）丙烯酰胺和1%（w/v）N, N’-亚甲双丙烯酰胺的贮存液，丙烯酰胺和双丙烯酰胺在贮存过程中缓慢转变为丙烯酸和双丙烯酸，这一脱氨基反应是光催化或碱催化的，故应核实溶液的pH值不超过7.0。这一溶液置棕色瓶中贮存于室温，每隔几个月须重新配制。 小心：丙烯酰胺和双丙烯酰胺具有很强的神经毒性并容易吸附于皮肤。 ② 十二烷基硫酸钠（SDS）。SDS可用去离子水配成10%（w/v）贮存液保存于室温。 ③用于制备分离胶和积层胶的Tris缓冲液。 ④TEMED(N,N,N’,N’-四甲基乙二胺)。TEMED通过催化过硫酸铵形成自由基而加速丙烯酰胺与双丙烯酰胺的聚合。 ⑤过硫酸铵。 过硫酸铵提供驱动丙烯酰胺和双丙烯酰胺聚合所必需的自由基。须新鲜配制。 ⑥Tris-甘氨酸电泳缓冲液。 ⑵ 装置： 使用不连续缓冲系统要求在垂直板凝胶上进行SDS聚丙烯酰胺电泳。 2．SDS聚丙烯酰胺凝胶的灌制 ⑴ 根据厂家说明书安装玻璃板。 ⑵ 确定所需凝胶溶液体积，按下表给出的数值在一小烧杯中按所需丙烯酰胺浓度配制一定体积的分离胶溶液。一旦加入TEMED，马上开始聚合，故应立即快速旋动混合物并进入下步操作。 配制Tris-甘氨酸SDS聚丙烯酰胺凝胶电泳分离胶溶液 溶液成分 总体积 5ml 总体积 10ml 总体积 15ml 总体积 20ml 总体积 25ml 总体积 30ml  6%        水 2.6ml 5.3ml 7.9ml 10.6ml 13.2ml 15.9ml  30%丙烯酰胺 1.0ml 2.0ml 3.0ml 4.0ml 5.0ml 6.0ml  1.5M Tris(pH 8.8) 1.3ml 2.5ml 3.8ml 5.0ml 6.3ml 7.5ml  10% SDS 0.05ml 0.1ml 0.15ml 0.2ml 0.25ml 0.3ml  10%过硫酸胺 0.05ml 0.1ml 0.15ml 0.2ml 0.25ml 0.3ml  TEMED 0.004ml 0.008ml 0.012ml 0.016ml 0.02ml 0.024ml  8%        水 2.3ml 4.6ml 6.9ml 9.3ml 11.5ml 13.9ml  30%丙烯酰胺 1.3ml 2.7ml 4.0ml 5.3ml 6.7ml 8.0ml  1.5M Tris(pH 8.8) 1.3ml 2.5ml 3.8ml 5.0ml 6.3ml 7.5ml  10% SDS 0.05ml 0.1ml 0.15ml 0.2ml 0.25ml 0.3ml  10%过硫酸胺 0.05ml 0.1ml 0.15ml 0.2ml 0.25ml 0.3ml  TEMED 0.003ml 0.006ml 0.009ml 0.012ml 0.015ml 0.018ml  10%        水 1.9ml 4.0ml 5.9ml 7.9ml 9.9ml 11.9ml  30%丙烯酰胺 1.7ml 3.3ml 5.0ml 6.7ml 8.3ml 10.0ml  1.5M Tris(pH 8.8) 1.3ml 2.5ml 3.8ml 5.0ml 6.3ml 7.5ml  10% SDS 0.05ml 0.1ml 0.15ml 0.2ml 0.25ml 0.3ml  10%过硫酸胺 0.05ml 0.1ml 0.15ml 0.2ml 0.25ml 0.3ml  TEMED 0.002ml 0.004ml 0.006ml 0.008ml 0.01ml 0.012ml  12%        水 1.6ml 3.3ml 4.9ml 6.6ml 8.2ml 9.9ml  30%丙烯酰胺 2.0ml 4.0ml 6.0ml 8.0ml 10.0ml 12.0ml  1.5M Tris(pH 8.8) 1.3ml 2.5ml 3.8ml 5.0ml 6.3ml 7.5ml  10% SDS 0.05ml 0.1ml 0.15ml 0.2ml 0.25ml 0.3ml  10%过硫酸胺 0.05ml 0.1ml 0.15ml 0.2ml 0.25ml 0.3ml  TEMED 0.002ml 0.004ml 0.006ml 0.008ml 0.01ml 0.012ml  15%        水 1.1ml 2.3ml 3.4ml 4.6ml 5.7ml 6.9ml  30%丙烯酰胺 2.5ml 5.0ml 7.5ml 10.0ml 12.5ml 15.0ml  1.5M Tris(pH 8.8) 1.3ml 2.5ml 3.8ml 5.0ml 6.3ml 7.5ml  10% SDS 0.05ml 0.1ml 0.15ml 0.2ml 0.25ml 0.3ml  10%过硫酸胺 0.05ml 0.1ml 0.15ml 0.2ml 0.25ml 0.3ml  TEMED 0.002ml 0.004ml 0.006ml 0.008ml 0.01ml 0.012ml   ⑶ 迅速在两玻璃板的间隙中灌注丙烯酰胺溶液,留出灌注浓缩胶所需空间(梳子的齿长再加0.5cm)。再在胶液面上小心注入一层水（约2~3mm高），以阻止氧气进入凝胶溶液。 ⑷ 分离胶聚合完全后（约30分钟），倾出覆盖水层，再用滤纸吸净残留水。 ⑸ 制备浓缩胶：按下表给出的数据，在另一小烧杯中制备一定体积及一定浓度的丙烯酰胺溶液，一旦加入TEMED，马上开始聚合，故应立即快速旋动混合物并进入下步操作。 配制Tris-甘氨酸 SDS聚丙烯酰胺凝胶电泳5% 浓缩胶溶液 溶液成分 总体积 3ml 总体积 4ml 总体积 5ml 总体积 6ml 总体积 8ml  水 2.1ml 2.7ml 3.4ml 4.1ml 5.5ml  30%丙烯酰胺 0.5ml 0.67ml 0.83ml 1.0ml 1.3ml  1M Tris(pH 6.8) 0.38ml 0.5ml 0.63ml 0.75ml 1.0ml  10% SDS 0.03ml 0.04ml 0.05ml 0.06ml 0.08ml  10%过硫酸胺 0.03ml 0.04ml 0.05ml 0.06ml 0.08ml  TEMED 0.003ml 0.004ml 0.005ml 0.006ml 0.008ml   ⑹聚合的分离胶上直接灌注浓缩胶,立即在浓缩胶溶液中插入干净的梳子。小心避免混入气泡，再加入浓缩胶溶液以充满梳子之间的空隙，将凝胶垂直放置于室温下。 ⑺在等待浓缩胶聚合时，可对样品进行处理，在样品中按1:1体积比加入样品处理液，在100℃加热3分钟以使蛋白质变性。 样品处理液配方： 50mM Tris-HCl(pH 6.8) 100mM DTT(or 5% 巯基乙醇) 2% SDS 0.1% 溴酚蓝 10%甘油 ⑻ 浓缩胶聚合完全后（30分钟），小心移出梳子。把凝胶固定于电泳装置上，上下槽各加入Tris-甘氨酸电极缓冲液。必须设法排出凝胶底部两玻璃板之间的气泡。 Tris-甘氨酸电极缓冲液： 25mM Tris 250mM 甘氨酸 (pH 8.3) 0.1% SDS ⑼ 按予定顺序加样，加样量通常为10～25μl（1.5mm厚的胶）。 ⑽ 将电泳装置与电源相接，凝胶上所加电压为8V/cm。当染料前沿进入分离胶后，把电压提高到15V/cm，继续电泳直至溴酚蓝到达分离胶底部上方约1cm，然后关闭电源。 ⑾ 从电泳装置上卸下玻璃板，用刮勺撬开玻璃板。紧靠最左边一孔（第一槽）凝胶下部切去一角以标注凝胶的方位。 3．用考马斯亮蓝对SDS聚丙烯酰胺凝胶进行染色 经SDS聚丙烯酰胺凝胶电泳分离的蛋白质样品可用考马斯亮蓝R250染色。 染色液： 0.1% 考马斯亮蓝 R250 40% 甲醇 10% 冰醋酸 染色1～2小时或过夜。 脱色液： 10% 甲醇 10% 冰醋酸 脱色需3～10小时，其间更换多次脱色液至背景清楚。 此方法检测灵敏度为0.2～1.0??g。脱色后，可将凝胶浸于水中，长期封装在塑料袋内而不降低染色强度。为永久性记录，可对凝胶进行拍照，或将凝胶干燥成胶片。 4. 测量并计算分子量 蛋白质的分子量与它的电泳迁移有一定关系式，经37种蛋白的测定得到以下的关系式： Mw ＝ K (10－bm) (1) lgMw = lg K －bm = K1－bm (2) 其中Mw是蛋白质分子量；K和K1为常数 b为斜率，m 是电泳迁移率，实际使用的是相对迁移率mR。 如果用几种标准蛋白质分子量的对数作纵坐标，用各自的相对迁移率作横坐标，即可画出一条斜率为负的标准曲线。相对迁移率为： 其中，dpr、dBPB分别为样品和BPB（溴酚兰）以分离胶表面为起点迁移的距离。 欲求未知蛋白的分子量，只需求出它的相对迁移率： mR未= dpr未/ dBPB 然后，从标准曲线上就可求出此未知蛋白的分子量。 取出脱色后的凝胶平放在两块透明投影胶片中间，赶尽气泡，在复印机上复印。在复印的凝胶图上用直尺分别量出各条蛋白带迁移的距离dpr和dBPB（以蛋白带的上沿或中心为准），计算相对迁移率，根据方程式： lgMw = K1－bmR 用各标准蛋白分子量的对数（纵坐标）和相对迁移率mR（横坐标）画出标准曲线，由标准曲线再求出其他各条待测和未知蛋白带的分子量，如有可能计算其误差。 SDS-PAGE I. BACKGROUND: Anionic surfactant compounds such as sodium dodecyl sulfate(SDS) are composed of a negatively charged ionic “head group” and a hydrophobic hydrocarbon “tail”. CH3CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 OSO3 – Na+ The high solubility in water imparted by the ionic head group and the high solubility in nonpolar solvents implied by the hydrocarbon tail result in a compromise in aqueous solution. Surfactant molecules form aggregates called micelles in aqueous solution. These aggregates satisfy the solubility characteristics of both the head and tail regions of the surfactant; ionic groups are exposed to water on the surface of the aggregate while the hydrophobic tails associate with each other within the interior of a roughly spherical aggregate of 60 to 100 molecules. Hydrophobic guest molecules can be taken into the interior of the micelle. This includes the hydrophobic amino acids normally confined to the interior of a native protein. The association between hydrophobic amino acid residues and the micelle interior is strong enough to denature most proteins, turning them inside-out so that become effectively coated with anionic surfactant molecules. Reductive cleavage of all –S-S- bonds followed by treatment with the anionic surfactant sodium dodecyl sulfate(SDS) will disrupt the native tertiary structure of most proteins, causing them to adopt rodlike structures. It has been established that this binding occurs with a constant surfactant-totprotein weight ratio and with enough anionic surfactants to totally dominate the native charge of the protein. Therefore, the charge per unit protein weight is nearly constant and the electrophoretic mobility of SDS denatured proteins is a function of size alone. The technique of SDS-polyacrylamide gel electrophoresis is widely used to determine the molecular weight of unknown proteins by comparing their relative electrophoretic mobility to standard proteins of known molecular weight. A direct comparison of the mobilities of known and unknown proteins run under identical conditions (in the same cell at the same time) is recommended. The mobilities of known proteins can be plotted as a function of log(molecular weight) and the mobilities of known proteins used to estimate molecular weights by extrapolation. A necessary first step in the protein sample preparation for SDS electrophoresis is treatment with an excess of 2-mercaptoethanol, which reduces all disulfide(-S-S-) bonds in the protein. This permits total disruption of the protein native structure, which is usually stabilized by disulfide linkages. Some proteins(e.g., chymotrypsin) contain polypeptides linked only by disulfide bonds. Reduction will yield two or more polypeptide fragments which will migrate independently. In the same way oligomeric proteins(e.g., hemoglobin) will dissociate into monomeric subunits when solubilized by SDS. For this reason, hemoglobin migrates as a monomer of molecular weight 16,000 rather than a tetramer of 64,500 D. The subunit dissociation caused by SDS is one reason why oligomeric proteins must be characterized by both SDS and disc electrophoresis. The pore size of the gel network is the critical factor in SDS-PAGE separations. The average pore size can be decreased by either increasing the total concentration of monomer(both acrylamide and BIS) or by increasing the proportion of cross-linker(BIS) to the total monomer in the gel. Table 1 gives the approximate molecular weight ranges that can be conveniently estimated with gels of various total monomer concentrations, expressed as percent-gel. %gel = (g Acrylamide + g BIS)/ 100ml x 100 Table 1. MOLECULAR WEIGHT RANGES FOR SDS-PAGE Total Acrylamide (% gel) 15 10 5  M.W. Range (Kilodaltons) 3-50 10-70 25-200   This methods separates proteins based primarily on their molecular weights (Laemmli, 1970). Among the varied uses of this technique are: Analysis of protein purity; Determination of protein molecular weight; Verification of protein concentration; Detection of proteolysis; Identification of immunoprecipitated proteins; First stage of immunoblotting; Detection of protein modification ; Separation and concentration of protein antigens for antibody production; Separation of radioactively labeled proteins. Sensitivity of staining: Coomassie Blue: 0.1~1 ?g per band (Smith, 1984); Silver Staining: 2~10 ng per band (Giulian et al., 1983). Time Required: Individual Steps: Pouring Separating Gel: 60 minutes Pouring Stacking Gel: 30 minutes Loading Samples: 15 minutes Electrophoresis: 45 minutes Staining: Coomassie Staining: 30 minutes (major bands), 3 hours for major bands to destain. Complete destaining may require 24-48 hours. Silver Staining: 6 hours. II. BUFFER SYSTEM: (A): Lower buffer: 18.17g Tris, 0.4g SDS, pH 8.8 (HCl), Added H2O to 100ml (1.5M Tris-HCl buffer). (B): Upper buffer: 6.06g Tris, 0.4g SDS, pH 6.8 (HCl), Added H2O to 100ml (0.5M Tris-HCl buffer). (C): 30% Acrylamide: 30g Acrylamide, 0.8g Bisacrylamide, Added H2O to 100ml. (D): 10%(w/v) Ammonium persulfate (fresh): 0.1g ammonium persulfate, Added H2O to 1ml. (F): Electrophoresis Buffer: 3g Tris, 14.4g glycine, 1g SDS, Added H2O to 1 liter. *H2O: Distilled water. *Amounts of Working Solutions to Use Volumes necessary for pouring gels of different thickness (for two 6x8cm gels) Gel Thickness Separating Stacking  0.5mm 5.6ml 1.4ml  0.75mm 8.4ml 2.1ml  1.0mm 11.2ml 2.8ml  1.5mm 16.8ml 4.2ml   Always prepare with a moderate excess of gel solution. Calculation for x% Separating Gel: Solution (C) x/3 ml Solution (A) 2.5 ml H2O (7.5-x/3) ml 10% Ammonium Persulfate 50??l TEMED 5??l **Total Volume 10 ml. *Pouring the Separating Gel Example of Separating Gel Preparation: 12.5% Running gel (Separating gel): A 4.5ml  C 7.5ml  H2O 5.9ml  TEMED 0.02ml   D(fresh) 0.07ml  Total 18ml  TEMED or D was added last. Assemble gel sandwich according to the manufacturer’s instructions, or according to the usage of alternative systems. For Mini-Gel, be sure that the bottom of both gel plates and spacers are perfectly flush against a flat surface before tightening clamp assembly. A slight misalignment will result in a leak. Combine solutions C and B and water in a small Erlenmeyer flask or test tube. Add ammonium persulfate and TEMED, and mix by swirling or inverting container gently (excessive aeration will interfere with polymerization). Work rapidly at this point because polymerization will be under way. Carefully introduce solution into gel sandwich using a pipet. Pipet solution so that it descends along a spacer. This minimizes the possibility of air bubbles becoming trapped with the gel. When the appropriate amount of separating gel solution has been added (in the case of the Mini-Gel, about 1.5cm from top of front plate or 0.5cm below level where teeth of comb will reach, Fig 1), gently layer about 1cm of water on top of the separating gel solution. This keeps the gel surface flat. Allow gel to polymerize (30-60 minutes). When the gel has polymerized, a distinct interface will appear between the separating gel and the water, and the gel mold can be tilted to verify polymerization. Fig 1. Separating gel prior to polymerization. *Pouring the Stacking Gel Example of Stacking Gel Preparation Stacking gel (3%): B 2.0ml  C 0.8ml  H2O 5.2ml  TEMED 0.01ml   D(fresh) 0.04ml  Total 8.05ml  TEMED or D was added last. Pour off water covering the separating gel. The small droplets remaining will not disturb the stacking gel. Combine Solution C and B and water in a small Erlenmeyer flask or a test tube. Add ammonium persulfate and TEMED and mix by gently swirling or inverting the container. Pipet stacking gel solution onto separating gel until solution reaches top of front plate. Carefully insert comb into gel sandwich until bottom of teeth reach top of front plate (Fig 2). Be sure no bubbles are trapped on ends of teeth. Tilting the comb at a slight angle is helpful for insertion without trapping air bubbles. Allow stacking gel to polymerize (about 30 minutes). After stacking gel has polymerized, remove comb carefully (making sure not to tear the well ears). Place gel into electrophoresis chamber. Add electrophoresis buffer to inner and outer reservoir, making sure that both top and bottom of gel are immersed in buffer. Fig 2. Inserting the sample-well comb into the stacking gel. III.PROTEIN SAMPLE PREPARATION: 1. Sample buffer: 1g SDS, 5ml Glycerol, 0.5mg Bromophenol blue, 2.5ml Mercaptoethanol, 10ml Upper buffer, Added H2O to 50ml. Protein sample is diluted with an equal volume of sample buffer. The mixture is heated in a boiling-water bath for 5 minutes. The amount of protein to be applied depends on the sample but applications in the range 1~5?g of protein per protein band usually give sharp, easily viewed bands. Sample volumes of 10~100?l are most common. *Capacity per Well (Mini-Gel System) Gel Thickness 1 Well 5 Wells 10 Wells 15 Wells  0.5mm 0.7ml 45?l 16?l 9?l  0.75mm 1.0ml 68?l 24?l 14?l  1.0mm 1.4ml 90?l 32?l 18?l  1.5mm 2.1ml 135?l 48?l 27?l   *Introduce sample solution into well using a Hamilton syringe (Fig 3). Layer protein solution on bottom of well and raise syringe tip as dye level rises. Be careful to avoid introducing air bubbles as this may allow some of sample to be carried to adjacent well. *Rinse syringe thoroughly with electrode buffer or water before loading different samples. Include molecular weight standards in one or both outside wells. Fig 3. Introducing protein solution into sample well. 2. Molecular Weight Standards: A slab gel is especially useful for molecular weight determinations since the sample and molecular weight standard proteins can be run under identical conditions on a single gel. There are a number of commercially available SDS-PAGE molecular weight standards which give a good spread of molecular weight lines in a gel. *Molecular Weight Standards Mid-Range Protein: Protein Molecular Weight(kD)  Phosphorylase b(Rabbit Muscle) 97.4  BSA(Bovine Serum Albumin) 66  Ovalbumin(Chicken Egg) 43  Carbonic anhydrase (Bovine Erythrocytes) 31  Lysozyme(Chicken Egg White) 14.4   IV. SLAB ELECTROPHORESIS CONDITIONS: Running a Gel *Steps Attach electrode plugs to proper electrodes. Current should flow towards the anode. Turn on power supply to 200V (constant voltage; amperage will be about 100mA at start, 60mA at end of electrophoresis for two 0.75mm gels; 110mA at start, 80mA at end for two 1.5mm gels). The dye front should migrate to 1cm from the bottom of the gel in 30-40 min for two 0.75mm gels (40-50 min for 1.5mm gels). *The high electrical current used in gel electrophoresis is very dangerous. Never disconnect electrodes before first turning off the power source. If using an electrophoresis apparatus which is not completely shielded from the environment, always leave a clearly visible sign warning that electrophoresis is in progress. Turn off power supply. Remove electrode plugs from electrodes. Remove gel plates from electrode assembly. Carefully remove a spacer, and inserting the spacer in one corner between the plates, gently pry apart the gel plates. The gel will stick to one of the plates. V. SDS-COOMASSIE STAINING AND DESTAINING: *Staining solution: 0.25g Coomassie Blue R-250 was dissolved in 50ml methanol, added 8ml acetic acid and 42ml H2O to 100ml. *Destaining solution: 200ml methanol, 70ml acetic acid, added H2O to 1000ml. The gels are stained in staining solution for 1~2hours, and are then soaked in destaining solution for 1~2hours, it is necessary to change fresh destaining solution several times. VI. GEL DRYING: The gels are first soaked in 30% Methanol/10%Glycerol solution for 30 minutes. It is carefully removed and sandwiched between a double layer of gel drying membrane on the drying frame(keep drying overnight).