【实验目的】
1.掌握葡聚糖凝胶的特性及凝胶层析的原理。 2．学习葡聚糖凝胶层析的基本操作技术。
【实验原理】
凝胶层析又称分子排阻层析或凝胶过滤，是以被分离物质的分子量差异为基础的一种层析分离技术，这一技术为纯化蛋白质等生物大分子提供了一种非常温和的分离方法。层析的固定相载体是凝胶颗粒，目前应用较广的是：具有各种孔径范围的葡聚糖凝胶（Sephadex）和琼脂糖凝胶（Sepharose）。
葡聚糖凝胶是由直链的葡聚糖分子和交联剂3—氯1，2—环氧丙烷交联而成的具有多孔网状结构的高分子化合物。凝胶颗粒中网孔的大小可通过调节葡聚糖和交联剂的比例来控制，交联度越大，网孔结构越紧密；交联度越小，网孔结构就越疏松，网孔的大小决定了被分离物质能够自由出入凝胶内部的分子量范围。可分离的分子量范围从几百到几十万不等。 葡聚糖凝胶层析，是使待分离物质通过葡聚糖凝胶层析柱，各个组分由于分子量不相同，在凝胶柱上受到的阻滞作用不同，而在层析柱中以不同的速度移动。分子量大于允许进入凝胶网孔范围的物质完全被凝胶排阻，不能进入凝胶颗粒内部，阻滞作用小，随着溶剂在凝胶颗粒之间流动，因此流程短，而先流出层析柱；分子量小的物质可完全进入凝胶颗粒的网孔内，阻滞作用大，流程延长，而最后从层析柱中流出。若被分离物的分子量介于完全排阻和完全进入网孔物质的分子量之间，则在两者之间从柱中流出，由此就可以达到分离目的。 本实验以葡聚糖凝胶G—25作为固定相载体，来分离蓝色葡聚糖—2000和溴酚蓝。蓝色葡聚糖—2000分子量接近2×106， 而溴酚蓝分子量为670，二者分子量相差较大，前者完全排阻，而后者则可完全进入凝胶颗粒网孔内，二者通过层析柱的时间不同而分开。
【实验材料】
1.实验器材 层析柱(1×20cm)附有一小段乳胶管及螺旋夹；洗脱液瓶(带下口的三角瓶，250m1)；试管及试管架；量筒10m1；721型分光光度计
2.实验试剂
(1) Tris—醋酸缓冲液(pH7.0):取0.0lmol/L Tris溶液(含0.1mol/L KCl)900ml，用浓醋酸调pH至7.0，加蒸馏水至1000m1。
(2) 溴酚蓝溶液：称取溴酚蓝10毫克，溶于5毫升乙醇中，充分搅拌使其溶解，然后逐滴加入Tris—醋酸缓冲液(pH7.0)至溶液呈深蓝色。
(3) 蓝色葡聚糖—2000溶液：称取蓝色葡聚糖—2000 10毫克，溶于2毫升Tris—醋酸缓冲液(pH7.0)中即成。
(4) 样品溶液：取溴酚蓝溶液0.1毫升，蓝色葡聚糖—2000溶液0.5毫升混匀后为上柱样品溶液。
(5)葡聚糖凝胶G-25 (Sephadex-G-25)
【实验操作】
1.实验凝胶的制备：商品凝胶是干燥的颗粒，使用时需经溶胀处理，称取4克葡聚糖凝胶G—25，加50毫升蒸馏水，搅拌均匀，在室温溶胀6小时，或沸水浴溶胀 2小时，一般采用后一种方法。再用倾泻法除去凝胶上层水及细小颗粒，用蒸馏水反复洗涤几次，再以缓冲溶液(pH7.0的Tris—醋酸溶液)洗涤2—3次，使pH和离子强度达到平衡，最后抽去溶液及凝胶颗粒内部气泡，凝胶可保存在缓冲液内。
2.装柱：将层析柱洗净，垂直固定在铁支架上，选择有薄膜端作为层析柱下口，将下口
接上乳胶管并用螺旋夹夹紧。层析柱中加入洗脱液，打开下口螺旋夹，让溶液流出，排除残留气泡，最后保留约2厘米高度的洗脱液，拧紧螺旋夹。将凝胶轻轻搅动均匀，用玻璃棒沿层析柱内壁缓缓注入柱中，待凝胶沉积到柱床下已超过l厘米时，打开下口螺旋夹，继续装柱至柱床高度达到8厘米,关闭出口。装柱过程中严禁产生气泡，尽可能一次装完，避免出现分层。再用洗脱液平衡l至2个柱床体积，凝胶面上始终保持有一定的洗脱液。平衡后，拧紧下端螺旋夹。
3.加样品：打开螺旋夹使柱面上的洗脱液流出，直至床面与液面刚好平齐为止，关闭下端出口。取溴酚蓝及蓝色葡聚糖—2000混合液0.3毫升，小心地加于凝胶表面上，切勿搅动层析柱床表面。打开下端出口，使样品溶液进入凝胶内，并开始收集流出液。当样品溶液恰好流至与凝胶表面平齐时，关闭下端出口。用少量洗脱液清洗层析柱加样区，共洗涤三次，每次清洗液应完全进入凝胶柱内后，再进行下一次洗涤。最后在凝胶表面上加入洗脱液，保持高度为3—4cm。
4.洗脱与收集：连接好凝胶柱层析系统，调节洗脱液流速为每分钟1毫升，进行洗脱。仔细观察样品在层析柱内的分离现象，收集洗脱液，每收集3毫升即换一支收集管(试管预先编号)，收集约20管左右，样品即可完全被洗脱下来。将各收集管中的洗脱液分别用721型分光光度计在波长540nm处测定其光密度。
5.凝胶回收处理方法：将样品完全洗脱下来后，继续用三倍柱床体积的洗脱液冲洗凝胶后，将柱下口放在小烧杯中，慢慢打开，再将上口慢慢松开，使凝胶全部回收至小烧杯中，备用。
【实验结果】
以洗脱管号为横坐标，以光密度为纵坐标作图即得洗脱曲线。分析曲线图并讨论实验结果。
【思考题】
1. 葡聚糖凝胶层析为何能分离不同的样品？ 2. 葡聚糖凝胶层析操作时应注意哪些问题？
Experiment 5. Sephadex Gel Chromatography
【Purpose】
1. Study the characterization of Sephadex gel and main principle of gel chromatography 2. Master the basic operational techniques of gel chromatography.
【Principle】
Gel filtration (also called molecular exclusive chromatography or gel sieve) is a kind of chromatography technique based on the difference of molecular weight and is one of the effective and mild methods extensively used to isolate and analyze the biomacromocular substances. The solid phase vector of this chromatography is gel particle, which has the effect of molecular sieve; at present the most widely used gel has various apertures, such as dextran gel (trade name: Sephadex), and agarose gel (tradename: Sepharose).
Sephadex gel is a kind of macromolecular compound composed of dextran of some molecular weigh dextrose - glycoside and chloroacetone and it has multiple reticulation structure. The gel of different \"mesh\" can be acquired by controlling the ratio of chloroacetone and dextran in cross- linking agent, and by controlling the condition of cross - link reaction to control the cross - linked degree. The higher the cross-link degree, the tighter the mesh structure, and vise versa. Mesh size determines the molecular range at which the isolated material could enter into the internal gel freely. By this means, the molecular weigh of the isolated material could range from several hundred to several hundred thousand.
Sephadex gel chromatography is based on the principle that when the substances being
isolated flow through the chromatography column, each component shifts at different rate because of the different molecular weight and the difference of obstruction in solid phase. The material molecular is larger than what can permissively enter into the gel mesh will be excluded completely; so it can not enter into the interior of gel particle, and the effect of exclusion is low; its course is short and shift rate is rapid, so it first flows out of the chromatography column with the solvent flowing between gel particles. On the contrary, the material of smaller molecular weigh penetrate into gel particle completely, so its effect of exclusion is high, its course is long and shift rate is slow, and as a result it flows out of the column later. If the molecular of the material is between that of completely excluded and that of completely penetrated, the material will flow out of the column between them. So we can isolate materials by this means.
In this experiment, we use Sephadex G-25 as phase vector to separate Blue Sephadex-2000 and bromophenol blue. The molecular weight of blue sephadex-2000 is approximately 2×106, while the molecular weight of bromophenol is about 670. Because of the obvious molecular difference, the former can be excluded completely and the latter can enter into the gel particle, so they can be separated by different elution time.
【Materials】
1. Apparatus
Chromatography column (1×20cm) with latex tubing and clips, A beaker of 100ml, Test tubes and test tube shelf, Measuring cylinder 10ml, 721 type spectrophotometer
2. Reagents
(1) Tris-Acetate buffer ( pH7.0)
Take 0.01mol/L Tris solution (contains 0.1mol/L KCl) 900ml, adjust pH value to 7.0 by acetate, then add distilled water to 1000ml.
(2) Bromophenol solution
Weigh 10mg bromophenol, dissolved in 5ml ethanol, stir to make it effectively solve, then add Tris-acetate buffer(pH7.0) gradually till the solution becomes dark blue.
(3) Blue sephadex-2000 solution:
Take blue sephadex-2000 10mg, dissolved in 2ml Tris-acetate buffer (pH7.0).
(4) Sample: Take the mixture of 0.1ml bromophenol and 0.5ml Blue sephadex-2000 (5) Sephadex G-25
【Procedures】
1. Gel preparation
The gel we buy is dry particles, so before using it, expand it. In this experiment, add 4g Sephadex G-25 to 50ml distilled water, stir, expand 6 hours at room temperature, or 2 hours in boiling, commonly we choose boiling. Remove the upper water and small particles with pour method, wash gel with distilled water several times, then wash it with tris-acetate buffer (pH7.0) several times to make the pH and ion concentration reach balance. Finally remove bubble in the gel and also solution, and then the gel can be preserved in the buffer.
2. Stuffing
Fix a clean chromatography column on the iron bracket vertically, choose the membrane end as the bottom part, add latex tube and clip it. Add eluent, open the exit to let the solution flow out, remove the bubbles, and then fix the clip when there is eluent about 2 cm higher than the gel surface. Stir the gel gently, add it with glassy stick along the inner wall of column, open the exit when the height of the gel deposited in the bottom of the column reaches 1cm, continue stuffing till the column bed reaches 10cm, shut off the exit. It should be ensured that there is no bubble or layers of the column bed in the process, or again. After filling, wash the column bed with eluent (about 1 to 2 column volume) and keep steady eluent on the surface of gel. After balancing it, shut off the exit.
3. Loading sample
Open the exit to make the eluent flow out till the bed surface meets the liquid surface, shut off the exit. Load 0.3ml sample, the mixture of bromophenol and blue sephadex-2000, carefully on the gel surface, do not stir the column bed surface. Open the exit to let the sample enter into the gel and begin to collect the eluent. When the sample solution flows to the surface of gel, shut off the exit. Wash the sampling region with little eluent three times and on each time, make sure the washing eluent flow completely into the gel. Finally ass eluent on the gel surface and keep the height of eluent 3-4cm higher than the gel surface.
4. Elution and Collection
Adjust the speeding of eluent to about 1ml/min. Observe the seperation process of the sample in the chromatography column, collect the eluent about 3ml per test tube, and number those test tubes. After collecting about 20 test tubes of eluent, the sample can be totally washed out. Inspect the absorbance with 721 type spectrophotometer under the wavelength 540nm of those eluent in each test tube.
5. Regeneration gel
After washing out the sample, wash the gel with the eluent about 3 times of column volume.
Put a glass beaker on the exit end, open bottom end and upper end slowly and respectively, then the gel flows into the beaker and can be used again.
【Results】
The number of test tube is x-axis, and the absorbance is y-axis. Draw the elution curve. Analysis the curve and discuss the experiment result.
【Questions】
1. What is the principle of separating components in mixture with Sephadex Gel Chromatography?
2. What are the attentions in Sephadex Gel Chromatography?
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