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1、实验四 蛋白质印迹分析【实验目的】了解蛋白质印迹法的基本原理及其操作和应用。【实验原理】蛋白质印迹法又称为免疫印迹法， 这是一种可以检测固定在固相载体上蛋白质的免疫化 学技术方法。待测蛋白既可以是粗提物也可以经过一定的分离和纯化 , 另外这项技术的应用 需要利用待测蛋白的单克隆或多克隆抗体进行识别。如图所示，可溶性抗原，也就是待测蛋白首先要根据其性质，如分子量，分子大小，电 荷以及其等电点等采用不同的电泳方法进行分离； 通过电流将凝胶中的蛋白质转移到聚偏二 氟乙烯膜上；利用抗体(一抗) 与抗原发生特异性结合的原理，以抗体作为探针钓取目的蛋 白。值得注意的是在加入一抗前应首先加入非特异性蛋白，
2、如牛血清白蛋白对膜进行 “封阻” 而防止抗体与膜的非特异性结合。经电泳分离后的蛋白往往需再利用电泳方法将蛋白质转移到固相载体上 , 我们把这个过 程称为电泳印迹。常用的两种电转移方法分别为 :1.半干法: 凝胶和固相载体被夹在用缓冲溶液浸湿的滤纸之间 ,通电时间为 10 分钟30 分钟。2. 湿法：凝胶和固相载体夹心浸放在转移缓冲溶液中，转移时间可从 45 分钟延长到过 夜进行。由于湿法的使用弹性更大并且没有明显浪费更多的时间和原料， 因此我们在这里只描述 湿法的基本操作过程。对于目的蛋白的识别需要采用能够识别一抗的第二抗体。 该抗体往往是购买的成品， 已 经被结合或标记了特定的试剂， 如辣根
3、过氧化物酶。 这种标记是利用辣根过氧化物酶所催化 的一个比色反应， 该反应的产物有特定的颜色且固定在固相载体上， 容易鉴别。 因此可通过 对二抗的识别而识别一抗， 进而判断出目标蛋白所在的位置。 其他的识别系统包括碱性磷酸 酶系统和 125I 标记系统。【实验材料】1. 实验器材SDS/PAGE实验相关材料；电转移装置；供电设备；PVDF膜 ( Millipore Immobion-P #IPVH000 10 ); Whatman 3MM纸；其他工具：镊子、海绵垫、剪子、手套、小塑料或玻璃容器、 浅盘。2. 实验试剂 10x 转移缓冲溶液(1L)： 30.3g Trizma base(0.25
4、M), 144 g 甘氨酸(1.92M),加蒸馏水至 1L, 此时 pH 约为 8.3，不必调整。1x转移缓冲溶液(2L):在1.4L蒸馏水中加入400 ml甲醇及200 ml10x转移缓冲 溶液。TBS 缓冲溶液：将1.22g Tris (10 mM)和8.78g NaCI(150 mM)加入到1L蒸馏水中，用HCl 调节 pH 至 7.5。 TTBS buffer :在 1L TBS 缓冲溶液中加入 0.5ml Tween 20 (0.05%)。 一抗：兔抗待测蛋白抗体(多克隆抗体) 。(6) 二抗：辣根过氧化物酶标记羊抗兔。3%封阻缓冲溶液(0.5L)：牛血清白蛋白15mg加入TBS缓冲
5、溶液并定容至 0.5L , 过滤，在 4C 保存以防止细菌污染。0.5%封阻缓冲溶液(0.5L):牛血清白蛋白2.5mg加入TTBS缓冲溶液并定容至 0.5L , 过滤，在4C保存以防止细菌污染。 显影试剂：1ml氯萘溶液(30mg/ml甲醇配置)，加入10 ml甲醇，加入TBS缓冲溶液 至 50 ml，加入 30 ul 30% H 2O2。 染色液：1g氨基黑18B (0.1%)，250ml异丙醇(25%)及100ml乙酸(10%)用蒸馏水定容 至1L。(11)脱色液：将350ml异丙醇(35%)和20 ml乙酸(2%)用蒸馏水定容至1L。电泳膜转移圭封闭和清洗E蛋白质印迹法基本操作过程【实
6、验操作】1. 蛋白质的分离根据目的蛋白的性质，利用电泳方法将其进行分离。为提高电转移的效率，通常采用 SDS/PAGE 技术。分离实验结束后，首先将样品墙的上边缘用小刀去除，然后在胶板的右上角切一个小口以便定位，小心放入转移缓冲溶液中待用。2. 电转移准备PVDF膜根据胶的大小剪出一片 PVDF膜，膜的大小应略微小于胶的大小。将膜置于甲醇中浸 泡1分钟，再移至转移缓冲溶液中待用。夹心放置顺序黑色筛孔板 海绵垫3MM PVDF 凝胶3MM纸海绵垫 白色筛孔板制作胶膜夹心在一浅盘中打开转移盒，将一个预先用转移缓冲溶液浸泡过的海绵垫放在转移盒的黑色 筛孔板上，在海绵垫的上方放置经转移缓冲溶液浸湿的3
7、MM纸，小心地将胶板放在 3MM纸上，并注意排除气泡。将PVDF膜放在胶的上方同时注意排除气泡，再在膜的上方放上一张同样用转移缓冲溶液浸湿过的3MM纸并赶出气泡，放置另一张浸泡过的海绵垫，关闭转移盒。将转移盒按照正确的方向放入转移槽中，转移盒的黑色筛孔板贴近转移槽的黑色端，转移盒的白色筛孔板贴近转移槽的白色端，填满转移缓冲溶液同时防止出现气泡。电转移连接电源，在4C条件下维持恒压100v，1小时。3.免疫检测膜染色断开电源，将转移盒从转移槽中移出，将转移盒的各个部分分开。用镊子将PVDF膜小心放入一个干净的容器中， 用TBS缓冲溶液进行短暂清洗， 从膜上剪下一条宽约 5mm的 膜放入另一个干净
8、的容器中。将这条膜在染色液中浸泡1分钟，然后在脱色液中脱色 30分钟，确定蛋白质已经转移到 PVDF膜上。膜的封闭和清洗对于没有进行染色的膜，首先倒出TBS缓冲溶液，加入3%封闭缓冲溶液，轻轻摇动至少1小时。倒掉3%封闭缓冲溶液，并用 TBS缓冲溶液清洗3次，每次5分钟。一抗倒掉TBS缓冲溶液，加入10 ml 0.5%封闭缓冲溶液及适量的一抗，轻轻摇动1小时以上。从容器中倒出一抗及封闭缓冲溶液，用TTBS缓冲溶液清洗两次，每次 10分钟。二抗倒出TTBS缓冲溶液，加入 5 ml 0.5%封闭缓冲溶液及适量的二抗。轻轻摇动30分钟，倒出二抗及封闭缓冲溶液，用TTBS缓冲溶液清洗两次，每次 10分
9、钟。检测倒掉TTBS缓冲溶液，并加入显影剂，轻轻摇动PVDF膜，观察显影情况，当能够清晰的看到显色带时，用蒸馏水在30分钟内分三次清洗 PVDF膜以终止显色反应的继续进行。【实验结果】检查膜上显色结果，蓝紫色带所对应的即是目标蛋白的位置。【思考题】1. 蛋白质印迹法的特点是什么？2. 请解释什么是BSA ?并说明它在本实验中的作用。3请说明二抗在蛋白质印迹法中的生物学功能。4.如何保存抗体？Experiment 16 Western Blot Analysis【 Purpose】Comprehend the theory of Western blotting; understand its
10、basic manipulation and application.【 Principle】Western blotting is also called Immunoblotting. It is a kind of immunochemical techniques which is used to detect a protein immobilized on a matrix. The target protein can be in a crude extract or a more purified preparation and the monoclonal or polycl
11、onal antibody against this protein is necessary to help us to recognize the antigen.As in the Figure, soluble antigens (the target protein) may be separated by electrophoresis based on its molecular weight (SDS/PAGE), size and charge (nondenaturating gel electrophoresis or isoelectric point (isoelec
12、tric focusing). After the separation, the proteins are transferred from the gel to a PVDF membrane. Once on the membrane antibodies (first antibodies) can be used to probe for the presence of particular protein because of the specifically binding of antigen with against it. Non-specific binding site
13、 can be “blocked ”using other non-specific protein such as bovine serum albumin before adding first antibody to avoid non-specific binding.Protein transfer is most commonly accomplished by electrophoresis ， This procedure is called electrophoretic blotting. The two common electrophoretic methods are
14、:1. Semi-dry blotting, in which the gel and immobilizing matrix are sandwiched between buffer-wetted filter papers through which a current is applied for 10-30 minutes.2. Wet (tank) blotting, in which the gel-matrix sandwich is submerged in transfer buffer for electrophoresis, which may take as litt
15、le as 45 minutes or may be allowed to continue overnightWe only describe wet blotting here, since it permits greater flexibility without being significantly more expensive in time or materials.The detection of target protein is using a second antibody, which can recognize the first antibody. Typical
16、ly, the second antibody is purchased already conjugated to a labeling agent such as the enzyme horseradish peroxidase. This marker is then visualized by a colorimetric reaction catalyzed by the enzyme which yields a colored product that remains fixed to the membrane. Thus, it is possible to recogniz
17、e first antibody through recognizing second antibody, and then identify125 the position of target protein. Other detection systems include alkaline phosphatase and I labels. 【 Materials 】1 Apparatus:Apparatus of SDS-PAGE, Electroblotting Apparatus, Power supply, PVDF membrane (Millipore Immobion-P #
18、IPVH 000 10), Whatman 3MM paper, Additional Tools: Forceps, sponge pad, scissor, gloves, small plastic or glass container, Shallow tray.2 Reagents: 10x transfer buffer (1 L): 30.3 g Trizma base (0.25 M), 144 g Glycine (1.92 M), pH shouldbe 8.3; without adjustment. 1x transfer buffer (2 L): 400 ml Me
19、thanol, 200 ml 10x transfer buffer, 1400 ml water. TBS buffer: Add 1.22g Tris (10 mM) and 8.78g NaCl(150 mM) to 1L distilled water and adjust pH to 7.5 with HCl. TTBS buffer: 1L TBS buffer add 0.5ml Tween 20 (0.05%). First antibody: antibody against the target protein. Second antibody: goat anti-rab
20、bit-HRP (horseradish peroxidase). 3% Blocking buffer (0.5 L): Add 15mg Bovine serum albumin in TBS buffer to final volume 0.5 L, keep at 4 C to pvent bacterial contamination. 0.5% Blocking buffer (0.5 L): Add 2.5mg Bovine serum albumin in TBS buffer to final volume0.5 L, keep at 4 C to prevent bacte
21、rial contamination.(9) Develop ing reage nt: 1ml chl ono aphthol solutio n (30mg/ml in metha nol), add 10 ml methanol, add TBS buffer to 50 ml and add 30 ul 30% H 2O2. Staining buffer: Add 1g amido black 18B (0.1%), 250ml isopropanol (25%) and 100 ml acetic acid (10%) to distilled water with final v
22、olume 1L.(11) Destaining buffer: Add 350ml isopropanol (35%) and 2 ml acetic acid(2%) to distilled water with final volume 1L.Blocki ng andAdditi on ofThe basic procedure of Wester n blott ing【Procedure】1. . Separation of ProteinRun an electrophoretic separation of known antigenic proteins. The meth
23、od of separation decided by the characters of target prote in, but for sufficie ntly tran sferri ng, the most com mon method is SDS-PAGE.After separation, remove upper side of sample wells with a razor blade. Notching bottom right-hand corner of gel for orientation and put gel in transfer buffer unt
24、il ready to use.2. . Electrotransfer(1) Preparation of membraneCut a piece of PVDF membra ne (Millipore Immobio n-P #IPVH 000 10) accordi ng to the size of gel. Incubate in methanol for about 1 min on a rocker at room temp. Remove methanol and equilibrate membra ne in 1x tran sfer buffer un til read
25、y to use. Arrange gel-membrane sandwichIn a shallow tray, ope n the tran sfer cassette. Put a well-soaked sponge pad on the black piece of the tran sfer cassette and a wetted 3MM paper on the sponge pad. Place the gel on the paper and arrange well so that all air bubbles are removed. Lay the PVDF me
26、mbrane on the top of gel and remove any air bubbles. Place a wetted sheet of 3MM paper over the PVDF membrane and remove the bubble. Covered with the sec ond well-soaked pad. Close the san dwich with the white piece of the cassette. Mount the san dwich in the tran sfer tank; put the black sides n ea
27、r the black side of the device. Fill the buffer tank with the tran sfer buffer. Cassette (black piece)l.*.I Sponge pad 3MM paper PVDF membranei i i i iii gel 3MM paperI.-1 Sponge pad Cassette (White piece)The arran geme nt of san dwich Electrotransfer:Attach the electrodes. Set the power supply to 1
28、00V (con sta nt voltage) for 1h at 4 C.3. . Immunodetection(1) Membrane stainingDisc onnect tran sfer apparatus, remove tran sfer cassette, and peel 3MM paper from membra ne. Remove the membra ne to a small container. Add 10 ml TBS buffer and wash for short time. Cut out one stripe with 5mm width an
29、d put in another clean container. Stain this stripe in staining buffer for 1 min. Destain for 30 min in destaining buffer to check whether protein has been tran sferred from gel to membra ne or n ot. Membrane blocking and washingFor other part of membrane, pour off TBS buffer. Add 3% blocking buffer
30、, rock gently for atleast 1 h. Pour off 3% block ing buffer and rinse briefly with TBS buffer three times, 5 minu tes for per time. First antibodyPour off TBS buffer. Add first antibody at appropriate dilution in 10 ml 0.5% blocking buffer. Rock gen tly for at least 1 h; pour off first an tibody sol
31、uti on from membra ne and wash twice for 10 mi nu tes with TTBS buffer. Second antibodyPour off TTBS buffer. Add sec ond an tibody at appropriate diluti on in 5 ml 0.5% block ing buffer. Rock gently for 30 min, pour off second antibody solution from membrane and wash twice for 10 mi nu tes with TTBS
32、 buffer. Detect ionPour off TTBS buffer from membra ne and add develop ing reage nt, Rock PVDF gen tly, monitoring development. When the bands can be seen clearly, stop development by washing membrane with distilled water for 30 minutes with 3 changes.【 Result】Check the bands on membrane, the band with blue-purple color corresponding to the target protein.【 Questions】1. What is the character of western blotting?2. What is the BSA? And explain its function during this experiment.3. Please elucidate the function of second antibody during western blotting.4. How to reserve antibody?
