实验六 SDS聚丙烯酰胺凝胶电泳法—蛋白质的分子量测定

【实验目的】

1.掌握SDS—聚丙烯酰胺电泳法的原理。

2.学会用此种方法测定蛋白质的分子量。

【实验原理】

SDS—聚丙烯酰胺凝胶电泳(SDS-PAGE)是对蛋白质进行量化，比较及特性鉴定的一种经济、快速、而且可重复的方法。该法主要依据蛋白质的分子量对其进行分离。SDS与蛋白质的疏水部分相结合，破坏其折叠结构，并使其稳定地存在于一个广泛均一的溶液中。SDS—蛋白质复合物的长度与其分子量成正比。由于在样品介质和聚丙烯酰胺凝胶中加入离子去污剂和强还原剂后，蛋白质亚基的电泳迁移率主要取决于亚基分子量的大小，而电荷因素可以被忽略。SDS—PAGE因易于操作和广泛的用途，使它成为许多研究领域中一种重要的分析技术。

SDS是十二烷基硫酸钠（sodium dodecyl sulfate）的简称，它是一种阴离子表面活性剂，加入到电泳系统中能使蛋白质的氢键和疏水键打开，并结合到蛋白质分子上（在一定条件下，大多数蛋白质与SDS的结合比为1.4gSDS/1g蛋白质），使各种蛋白质—SDS复合物都带上相同密度的负电荷，其数量远远超过了蛋白质分子原有的电荷量，从而掩盖了不同种类蛋白质间原有的电荷差别。这样就使电泳迁移率只取决于分子大小这一因素，于是根据标准蛋白质分子量的对数和迁移率所作的标准曲线，可求得未知物的分子量。

【实验材料】

1.实验器材

微型凝胶电泳装置；电源（电压200V，电流500mA）；100℃沸水浴；Eppendorf管；微量注射器（50μl或100μl）；干胶器、真空泵或水泵；带盖的玻璃或塑料小容器；摇床。

2.实验试剂

⑴ 2mol/L Tris-HCl (pH8.8):取24.2g Tris, 加50ml蒸馏水，缓慢的加浓盐酸至pH8.8（约加4ml）；让溶液冷却至室温，pH将会升高，加蒸馏水至100ml。

⑵ 1mol/L Tris-HCl (pH8.8):取12.1g Tris, 加50ml蒸馏水，缓慢的加浓盐酸至pH6.8（约加8ml）；让溶液冷却至室温，pH将会升高，加蒸馏水至100ml。

⑶ 10% (w/v) SDS: 取10g的SDS，加蒸馏水至100ml。

⑷ 50% (v/v) 甘油: 取50ml 100%甘油，加入50ml蒸馏水。

⑸ 1% (w/v) 溴酚蓝:取100mg溴酚蓝，加蒸馏水至10ml，搅拌,直到完全溶解，过滤除去聚合的染料。

⑹ A液--丙烯酰胺储备液（配制含30% (w/v) 丙烯酰胺和0.8% (w/v) 甲叉双丙烯酰胺的溶液100ml）在通风柜中操作，取29.2g丙烯酰胺，0.8g甲叉双丙烯酰胺，加蒸馏水至100ml，缓慢搅拌直至丙烯酰胺粉末完全溶解，用石蜡膜封口，可在4℃存放数月。

⑺ B液--4×分离胶缓冲液：取75ml 2mol/L Tris-HCl (pH8.8)，加入4ml 10% SDS, 加21ml蒸馏水，混匀，可在4℃存放数月。

⑻ C液--4×浓缩胶缓冲液:取50ml 1mol/L Tris-HCl (pH6.8)，加入4ml 10% SDS, 加46ml蒸馏水，混匀，可在4℃存放数月。

⑼ 10%过硫酸铵：取0.5g过硫酸铵，加入5ml蒸馏水，可保存在密封的管内，于4℃存放数月。

⑽ 电泳缓冲液：取3g Tris，14.4g甘氨酸，1g SDS，加蒸馏水至1L, pH约为8.3, 也可配制成10×的储备液，在室温下长期保存。

⑾ 5×样品缓冲液：取0.6ml 1mol/L Tris-HCl (pH6.8)，加入2ml 10% SDS, 5ml 50%的甘油，0.5ml 2-巯基乙醇，1ml 1%溴酚蓝，0.9ml的蒸馏水混匀，可在4℃保存数周，或在-20℃保存数月。

⑿ 考马斯亮蓝染液：1.0g 考马斯亮蓝R-250，加入450ml甲醇，450ml蒸馏水及100ml冰醋酸即成。

⒀ 考马斯亮蓝脱色液：将100ml甲醇，100ml冰醋酸，800ml蒸馏水混匀备用。

【实验操作】

1.灌制分离胶

⑴ 组装凝胶模具: 可按照使用说明书装配好灌胶用的模具。对于Bio-Rad的微型凝胶电泳系统，在上紧螺丝之前，必须确保凝胶玻璃板和隔片的底部与一个平滑的表面紧密接触，有细微的不匹配就会导致凝胶的渗漏。

⑵ 将A液、B液及蒸馏水在一个小烧瓶或试管中混合，丙烯酰胺(A液中)是神经毒素，操作时必须戴手套。加入过硫酸铵和TEMED后，轻轻搅拌使其混匀(过量气泡的产生会干扰聚合)。凝胶很快会聚合，操作要迅速。小心将凝胶溶液用吸管沿隔片缓慢加入模具内，这样可以避免在凝胶内产生气泡。

⑶ 当加入适量的分离胶溶液时(对于小凝胶，凝胶液加至约距前玻璃板顶端1.5cm或距梳子齿约0.5cm)，轻轻在分离胶溶液上覆盖一层1mm～5mm的水层，这使凝胶表面变得平整。当凝胶聚合后，在分离胶和水层之间将会出现一个清晰的界面。

2.灌制浓缩胶

⑴ 吸尽覆盖在分离胶上的水后将A液、C液和蒸馏水在三角烧瓶或小试管中混合。加入过硫酸铵和TEMED，并轻轻搅拌使其混匀。

⑵ 将浓缩胶溶液用吸管加至分离胶的上面，直至凝胶溶液到达前玻璃板的顶端。将梳子插入凝胶内，直至梳子齿的底部与前玻璃板的顶端平齐。必须确保梳子齿的末端没有气泡。将梳子稍微倾斜插入可以减少气泡的产生。

⑶ 凝胶聚合后，小心拔出梳子，不要将加样孔撕裂。将凝胶放入电泳槽内，如果使用Bio-Rad的微型凝胶系统，可预先接好电极。将电泳缓冲液加入内外电泳槽中，使凝胶的上下端均能浸泡在缓冲液中。

3.制备样品和上样

⑴ 将蛋白质样品与5x样品缓冲液(20μl+5μl)在一个Eppendorf管中混合。100℃加热2 min～10min。离心1s，如果有大量蛋白质碎片则应延长离心时间。

⑵ 用微量注射器将样品加入样品孔中。将蛋白质样品加至样品孔的底部，并随着染料水平的升高而升高注射器针头。避免带入气泡，气泡易使样品混入到相邻的加样孔中。

4.电泳

⑴ 将电极插头与适当的电极相接。电流流向阳极。将电压调至200V（保持恒压；对于两块0.75mm的胶来说，电流开始时为100mA，在电泳结束时应为60mA；对于两块1.5mm的胶来说，开始时应为110mA，结束时应为80mA。）。

⑵ 对于两块0.75mm的凝胶，染料的前沿迁移至凝胶的底部约需30～40分钟(1.5mm的凝胶则需40 min～50min)。关闭电源,从电极上拔掉电极插头,取出凝胶玻璃板,小心移动两玻璃板之间的隔片，将其插入两块玻璃板的一角。轻轻撬开玻璃板，凝胶便会贴在其中的一块板上。

5.考马斯亮蓝染色

这种染色方法在单条电泳带中蛋白质最小检出量为0.1μg的蛋白。通常可以根据所需要的敏感度来选择是使用考马斯亮蓝染色或银染色。

⑴ 戴上手套避免将手指印留在电泳凝胶上，将凝胶移入一个小的盛有少量考马斯亮蓝(20ml已经足够)的容器内(小心不要将胶撕破)。或将玻璃板连同凝胶浸在染料中轻轻振荡直至凝胶脱落。

⑵ 对于0.75mm的凝胶，可在摇床上缓慢震荡5分钟~l0分钟，对于1.5mm的凝胶，则需10分钟～20分钟，在染色和脱色过程中要用盖子或封口膜密闭容器口。弃去染液，将凝胶在水中漂洗数次。戴手套以避免将双手染色。

⑶ 加入考马斯亮蓝脱色液(约50ml)，清晰的条带很快会显现出来，大部分凝胶脱色需要1h，使用过的脱色液则可用水冲洗掉。为了脱色完全，需数次更换脱色液并震荡过夜。

6.干胶

⑴ 用一张l0cm×l2cm的Whatman 3MM滤纸覆盖凝胶，用一张玻璃纸或塑料保鲜膜覆盖在凝胶的另一个表面，小心不要将气泡裹进去，这样会导致凝胶的破裂。可用一个试管作为卷轴推赶，可以有效的除去气泡。

⑵ 将滤纸置于干胶器上，开启加热和抽真空开关，并盖上带有密封圈的盖子。待凝胶烘干后小心取出即可。

【实验结果】

根据凝胶中标准品与待测样品的相对迁移率判断待测样品的大致分子量。

【思考题】

⒈ 利用SDS—聚丙烯酰胺电泳法测定蛋白质的分子量与利用凝胶层析测定蛋白质的分子量有何不同？

⒉ SDS在该电泳方法中的作用是什么？

Experiment 6 SDS-polyacrylamide Gel Electrophoresis

—Assay Molecular Weight of Protein

【Purpose】

1 Master the principle of SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).

2 Learn to use this approach to determine the molecular weight of protein.

【Principle】

SDS is the short term of sodium dodecyl sulfate. It is a kind of anion surface-active agent. It can open the hydrogen bond and hydrophobic bond in the electrophoresis system and combine with the protein molecule (under a certain condition, most of proteins combine with SDS in a ratio of 1.4 gram of SDS per gram protein). As a result, all kinds of SDS-protein complexes carry the same negative charge density that is far more than the native net charge of the protein. In this case, the difference of charge between different kinds of proteins will be covered. Thus the electrophoretic mobility only depends on the molecular size. And according to the standard curve of the log molecular weight of standard proteins and the electrophoretic mobility, the molecular weight of unknown proteins can be estimated.

【Materials】

1 Apparatus

Minigel apparatus; Power supply (capacity 200V, 500MA); Boiling water bath; Eppendorf centrifuge (optional); Hamilton Syringes (50μl and 100μl capacity); Gel dryer and high vacuum pump or water pump (optional); Small glass or plastic container with lid (i.e. 12 cm×16 cm×3cm); Rocking or rotary shaker.

2 Reagents

⑴ 2M Tris-HCl (pH 8.8), 100ml: Weigh out 24.2g Tris, add to 50ml distilled water and add concentrated HCI slowly to pH 8.8 (about 4ml) (allow solution to cool to room temperature, pH will increase), add distilled water to a total volume of 100ml;

⑵ 1M Tris-HCl (pH 6.8), 100ml: Weigh out 12.1g Tris, add to 50ml distilled water and add concentrated HCI slowly to pH 6.8 (about 8ml) (allow solution to cool to room temperature, pH will increase), add distilled water to a total volume of 100ml;

⑶ 10% (w/v) SDS, 100ml: Weigh out 10g SDS and add distilled water to a total volume of 100ml, then store at room temperature;

⑷ 50% (v/v) glycerol, l00ml: Pour 50ml 100% glycerol and add 50ml distilled water;

⑸ 1% (w/v) bromophenol blue, 10ml: Weigh out 100mg bromophenol blue, add 10ml distilled water and stir until bromophenol blue dissolved, then filtration will remove aggregated dye.

⑹ Solution A (Acrylamide Stock Solution), 30% (w/v) acrylamide, 0.8% (w/v) bis-acrylamide, 100ml: Weigh out 29.2g acrylamide and 0.8g bis-acrylamide, add distilled water to make 100ml and stir until completely dissolved. Work under hood and keep acrylamide solution covered with Parafilm until acrylamide powder is completely dissolved, it can be stored for months in the refrigerator.

⑺ Solution B (4x Separating Gel Buffer), 100ml: Mix 75ml 2M Tris-HCl (pH 8.8), 4ml 10% SDS and 21ml H20 together, it can be stored for months in the refrigerator.

⑻ Solution C (4x Stacking Gel Buffer), 100ml: Mix 50ml 1M Tris-HCl (pH 6.8), 4ml 10% SDS and 46ml H20 together, it can be stored for months in the refrigerator.

⑼ 10% ammonium persulfate, 5ml: Weigh out 0.5g ammonium persulfate and 5ml H2O, it can be stored for months in a capped tube in the refrigerator;

⑽ Electrophoresis Buffer，1L: Weigh out 3g Tris, 14.4g glycine, lg SDS and add H20 to make 1L, pH should be approximately 8.3. It also can be make a 10×stock solution, stable indefinitely at room temperature;

⑾ 5x Sample Bufler，10ml: Mix 0.6ml 1M Tris-HCl (pH 6.8), 5ml 50% glycerol, 2ml 10% SDS, 0.5ml 2-mercaptoethanol, 1ml 1% bromophenol blue and 0.9ml H2O together, it can be stable for weeks in the refrigerator or for months at -20°C;

⑿ Coomassie Gel Stain, 1 L: Add 1.0g Coomassie Blue R-250 into 450ml methanol, 450ml H20 and 100ml glacial acetic acid;

⒀ Coomassie Gel Destain, 1 L: Mix 100ml methanol, 100ml glacial acidic acid and 800ml H20 together.

【Procedures】

1 Pouring the Separating Gel

⑴ Assemble gel sandwich according to the manufacturer's instructions, in the case of commercial apparatus (e.g., Bio-Rad Mini-Gel), 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.

⑵ Combine Solutions A and B and water in a small Erlenmeyer flask or a nest tube. Acrylamide (in Solution A) is a neurotoxin, so plastic gloves should be worn at all times; 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 descending along a spacer by using a pipet. This minimizes the possibility of air bubbles becoming trapped within the gel.

⑶ 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,), gently layer about l cm of water on top of the separating gel solution. This keeps the gel surface flat. When the gel has polymerized, a distinct interface will appear between the separating gel and the water.

⒉. Pouring the Stacking Gel

⑴ Pour off water covering the separating gel. The small droplets remaining will not disturb the stacking gel; Combine Solutions A and C 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.

⑵ Pipet stacking gel solution onto separating gel until solution reaches top of front plate; Carefully insert comb into gel sandwich until bottom of teeth reaches top of front plate. 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;

⑶ After stacking gel has polymerized, remove comb carefully (making sure not to tear the well ears); Place gel into electrophoresis chamber. If using the Mini-Gel system, attach both gels to electrode assembly before inserting into electrophoresis tank; Add electrophoresis buffer to inner and outer reservoir, making sure that both top and bottom of gel are immersed in buffer.

3 Preparing and Loading Samples

⑴ Combine protein sample with 5×Sample Buffer (i.e.20µl+5µl) in an Eppendorf tube; Heat at 100℃ for 2 minutes～10 minutes; Spin down protein solution for 1 second in microfuge-longer if large quantities of debris are present.

⑵ Introduce sample solution into well using a Hamilton syringe. 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.

4 Running a Gel

⑴ 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 gets);

⑵ The dye front should migrate to lcm from the bottom of the gel in 30 min～40 min for two 0.75mm gels (40 min～50 min for 1.5mm gels); Turn off power supply; Remove electrode plugs from electrodes; Remove gel plates from electrode assembly; Carefully remove a spacer, and insert the spacer in one corner between the plates, gently pry apart the gel plates. The gel will stick to one of the plates.

5 Staining a Gel with Coomassie Blue

This method of staining can detect as little as 0.lμg of protein in a single band. Generally a choice is made between using Coomassie Blue or Silver Stain depending on sensitivity desired.

⑴ Wearing gloves to prevent transfer of fingerprints to the gel, pick up the gel and transfer it to a small container (taking care not to tear the gel) containing a small amount of Coomassie Stain (20ml is sufficient), or gently agitate the glass plate in stain solution until gel separates from plate;

⑵ Agitate for 5 minutes～10 minutes for 0.75mm, 10 minutes～20 minutes for 1.5mm gel on slow rotary or rocking shaker. Cover container with lid or plastic wrap during staining and destaining; Pour out stain (can be reused several times, but it is fairly inexpensive so we generally discard it) and rinse the gel with a few changes of water. Use gloves to avoid staining hands;

⑶ Add destaining buffer (about 50ml). Strong bands are visible immediately on a light box, and the gel is largely destained within an hour. Used destaining buffer can be washed down the sink with ample water. To destain completely, change destain solution and agitate overnight.

6 Drying a Gel

⑴ Cover gel with a 10 cm×12 cm piece of Whatman 3MM paper and the other side of gel is covered with an acetate sheet or plastic wrap taking care not to trap air bubbles, which can lead to gel cracking. It is useful to roll out air bubbles, using a test tube as a rolling pin;

⑵ Place Whatman paper on gel dryer, turn on heat and suction, and cover with sealing gasket. Drying can be finished when the gel has been dried.

【Result】

According to the electrophoretic mobility of the standard protein and the unknown protein, identify the putative molecular weight of unknown protein.

【Questions】

1. What is the difference of molecular weight assay between SDS/PAGE and gel chromatography?

2. What is the biological function of SDS in SDS/PAGE?