蛋白质印迹分析（Western Blot Analysis）

【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，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。

【实验操作】

⒈.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。

⒉.电转移

⑴ 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
⑵ 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3MM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。
⑶ 电转移
连接电源,在4°C条件下维持恒压100v，1小时
⒊.免疫检测
⑴ 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。
⑵ 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。
⑶ 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。
⑷ 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分钟。
⑸ 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。
                     Western Blot Analysis
 
【Purpose】
Comprehend the theory of Western blotting; understand its 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 polyclonal 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 （isoelectric 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 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:
⒈ 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.
⒉ Wet （tank） blotting, in which the gel-matrix sandwich is submerged in transfer buffer for electrophoresis, which may take as little as 45 minutes or may be allowed to continue overnight
We 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. Typically, 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 recognize first antibody through recognizing second antibody, and then identify the position of target protein. Other detection systems include alkaline phosphatase and 125I labels.
【Materials】
⒈ Apparatus:
Apparatus of SDS-PAGE, Electroblotting Apparatus, Power supply, PVDF membrane（Millipore Immobion-P #IPVH 000 10）, Whatman 3MM paper, Additional Tools: Forceps, sponge pad, scissor, gloves, small plastic or glass container, Shallow tray.
⒉ Reagents:
⑴ 10x transfer buffer （1 L）: 30.3 g Trizma base （0.25 M）, 144 g Glycine （1.92 M）, pH should be 8.3; without adjustment.
⑵ 1x transfer buffer （2 L）: 400 ml Methanol, 200 ml 10x transfer buffer, 1400 ml water.
⑶ 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.
⑷ TTBS buffer: 1L TBS buffer add 0.5ml Tween 20 （0.05%）.
⑸ First antibody: antibody against the target protein.
⑹ Second antibody: goat anti-rabbit-HRP（horseradish peroxidase）.
⑺ 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 prevent bacterial contamination.
⑻ 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 bacterial contamination.
⑼ Developing reagent: 1ml chlonoaphthol solution （30mg/ml in methanol）, add 10 ml methanol, add TBS buffer to 50 ml and add 30 ul 30% H2O2.
⑽ Staining buffer: Add 1g amido black 18B （0.1%）, 250ml isopropanol （25%） and 100 ml acetic acid （10%） to distilled water with final volume 1L.
⑾ Destaining buffer: Add 350ml isopropanol （35%） and 2 ml acetic acid（2%） to distilled water with final volume 1L.

【Procedure】
⒈. Separation of Protein
Run an electrophoretic separation of known antigenic proteins. The method of separation decided by the characters of target protein, but for sufficiently transferring, the most common 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 until ready to use.
⒉. Electrotransfer
⑴ Preparation of membrane 
Cut a piece of PVDF membrane （Millipore Immobion-P #IPVH 000 10） according to the size of gel. Incubate in methanol for about 1 min on a rocker at room temp. Remove methanol and equilibrate membrane in 1x transfer buffer until ready to use.
⑵ Arrange gel-membrane sandwich
In a shallow tray, open the transfer cassette. Put a well-soaked sponge pad on the black piece of the transfer 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 membrane 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 second well-soaked pad. Close the sandwich with the white piece of the cassette. Mount the sandwich in the transfer tank; put the black sides near the black side of the device. Fill the buffer tank with the transfer buffer.

⑶ Electrotransfer:
Attach the electrodes. Set the power supply to 100V （constant voltage） for 1h at 4°C.
⒊. Immunodetection
⑴ Membrane staining
Disconnect transfer apparatus, remove transfer cassette, and peel 3MM paper from membrane. Remove the membrane to a small container. Add 10 ml TBS buffer and wash for short time. Cut out one stripe with 5mm width and 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 transferred from gel to membrane or not.
⑵ Membrane blocking and washing
For other part of membrane, pour off TBS buffer. Add 3% blocking buffer，rock gently for at least 1 h. Pour off 3% blocking buffer and rinse briefly with TBS buffer three times, 5 minutes for per time.
⑶ First antibody
Pour off TBS buffer. Add first antibody at appropriate dilution in 10 ml 0.5% blocking buffer. Rock gently for at least 1 h; pour off first antibody solution from membrane and wash twice for 10 minutes with TTBS buffer.
⑷ Second antibody
Pour off TTBS buffer. Add second antibody at appropriate dilution in 5 ml 0.5% blocking buffer. Rock gently for 30 min, pour off second antibody solution from membrane and wash twice for 10 minutes with TTBS buffer.
⑸ Detection
Pour off TTBS buffer from membrane and add developing reagent, Rock PVDF gently, 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.

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 #IPVH 000 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。

⑴ 10x转移缓冲溶液（1L）：30.3g Trizma base（0.25M）, 144 g甘氨酸（1.92M）,加蒸馏水至1L, 此时pH约为8.3，不必调整。

⑵ 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。

⑶ TBS 缓冲溶液:将1.22g Tris （10 mM）和8.78g NaCl（150 mM）加入到1L蒸馏水中，用HCl调节pH至7.5。

⑷ TTBS buffer：在1L TBS 缓冲溶液中加入0.5ml Tween 20（0.05%）。

⑸ 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。

⑺ 3% 封阻缓冲溶液（0.5L）：牛血清白蛋白15mg加入TBS缓冲溶液并定容至0.5L，过滤，在4°C 保存以防止细菌污染。

⑻ 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4°C 保存以防止细菌污染。

⑼ 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% H₂O₂。

⑽ 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。

⑾ 脱色液：将350ml异丙醇（35%）和20 ml乙酸（2%）用蒸馏水定容至1L。