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蛋白質(zhì)印跡分析（Western Blot Analysis）

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蛋白質(zhì)印跡分析（Western Blot Analysis）

【實驗?zāi)康摹?/span>
了解蛋白質(zhì)印跡法的基本原理及其操作和應(yīng)用。

【實驗原理】
蛋白質(zhì)印跡法又稱為免疫印跡法，這是一種可以檢測固定在固相載體上蛋白質(zhì)的免疫化學(xué)技術(shù)方法。待測蛋白既可以是粗提物也可以經(jīng)過一定的分離和純化,另外這項技術(shù)的應(yīng)用需要利用待測蛋白的單克隆或多克隆抗體進行識別。
如圖所示，可溶性抗原，也就是待測蛋白首先要根據(jù)其性質(zhì)，如分子量，分子大小，電荷以及其等電點等采用不同的電泳方法進行分離；通過電流將凝膠中的蛋白質(zhì)轉(zhuǎn)移到聚偏二氟乙烯膜上；利用抗體（一抗）與抗原發(fā)生特異性結(jié)合的原理，以抗體作為探針釣取目的蛋白。值得注意的是在加入一抗前應(yīng)首先加入非特異性蛋白，如牛血清白蛋白對膜進行“封阻”而防止抗體與膜的非特異性結(jié)合。
經(jīng)電泳分離后的蛋白往往需再利用電泳方法將蛋白質(zhì)轉(zhuǎn)移到固相載體上,我們把這個過程稱為電泳印跡。常用的兩種電轉(zhuǎn)移方法分別為:
1.半干法: 凝膠和固相載體被夾在用緩沖溶液浸濕的濾紙之間,通電時間為10分鐘~30分鐘。
2.濕法：凝膠和固相載體夾心浸放在轉(zhuǎn)移緩沖溶液中，轉(zhuǎn)移時間可從45分鐘延長到過夜進行。
由于濕法的使用彈性更大并且沒有明顯浪費更多的時間和原料，因此我們在這里只描述濕法的基本操作過程。
對于目的蛋白的識別需要采用能夠識別一抗的第二抗體。該抗體往往是購買的成品，已經(jīng)被結(jié)合或標記了特定的試劑，如辣根過氧化物酶。這種標記是利用辣根過氧化物酶所催化的一個比色反應(yīng)，該反應(yīng)的產(chǎn)物有特定的顏色且固定在固相載體上，容易鑒別。因此可通過對二抗的識別而識別一抗，進而判斷出目標蛋白所在的位置。其他的識別系統(tǒng)包括堿性磷酸酶系統(tǒng)和¹²⁵I標記系統(tǒng)。

【實驗操作】

⒈.蛋白質(zhì)的分離

根據(jù)目的蛋白的性質(zhì)，利用電泳方法將其進行分離。為提高電轉(zhuǎn)移的效率，通常采用SDS/PAGE技術(shù)。
分離實驗結(jié)束后，首先將樣品墻的上邊緣用小刀去除，然后在膠板的右上角切一個小口以便定位，小心放入轉(zhuǎn)移緩沖溶液中待用。

⒉.電轉(zhuǎn)移

⑴ 準備PVDF膜
根據(jù)膠的大小剪出一片PVDF膜，膜的大小應(yīng)略微小于膠的大小。將膜置于甲醇中浸泡1分鐘，再移至轉(zhuǎn)移緩沖溶液中待用。

夾心放置順序
⑵ 制作膠膜夾心
在一淺盤中打開轉(zhuǎn)移盒，將一個預(yù)先用轉(zhuǎn)移緩沖溶液浸泡過的海綿墊放在轉(zhuǎn)移盒的黑色篩孔板上，在海綿墊的上方放置經(jīng)轉(zhuǎn)移緩沖溶液浸濕的3MM紙，小心地將膠板放在3MM紙上，并注意排除氣泡。將PVDF膜放在膠的上方同時注意排除氣泡，再在膜的上方放上一張同樣用轉(zhuǎn)移緩沖溶液浸濕過的3MM紙并趕出氣泡，放置另一張浸泡過的海綿墊，關(guān)閉轉(zhuǎn)移盒。將轉(zhuǎn)移盒按照正確的方向放入轉(zhuǎn)移槽中，轉(zhuǎn)移盒的黑色篩孔板貼近轉(zhuǎn)移槽的黑色端，轉(zhuǎn)移盒的白色篩孔板貼近轉(zhuǎn)移槽的白色端，填滿轉(zhuǎn)移緩沖溶液同時防止出現(xiàn)氣泡。
⑶ 電轉(zhuǎn)移
連接電源,在4°C條件下維持恒壓100v，1小時
⒊.免疫檢測
⑴ 膜染色
斷開電源，將轉(zhuǎn)移盒從轉(zhuǎn)移槽中移出，將轉(zhuǎn)移盒的各個部分分開。用鑷子將PVDF膜小心放入一個干凈的容器中，用TBS緩沖溶液進行短暫清洗，從膜上剪下一條寬約5mm的膜放入另一個干凈的容器中。將這條膜在染色液中浸泡1分鐘，然后在脫色液中脫色30分鐘,確定蛋白質(zhì)已經(jīng)轉(zhuǎn)移到PVDF膜上。
⑵ 膜的封閉和清洗
對于沒有進行染色的膜，首先倒出TBS緩沖溶液,加入3%封閉緩沖溶液,輕輕搖動至少1小時。倒掉3%封閉緩沖溶液，并用TBS緩沖溶液清洗3次, 每次5分鐘。
⑶ 一抗
倒掉TBS緩沖溶液，加入10 ml 0.5%封閉緩沖溶液及適量的一抗，輕輕搖動1小時以上。從容器中倒出一抗及封閉緩沖溶液，用TTBS緩沖溶液清洗兩次，每次10分鐘。
⑷ 二抗
倒出TTBS 緩沖溶液，加入5 ml 0.5%封閉緩沖溶液及適量的二抗。輕輕搖動30分鐘，倒出二抗及封閉緩沖溶液，用TTBS緩沖溶液清洗兩次，每次10分鐘。
⑸ 檢測
倒掉TTBS 緩沖溶液，并加入顯影劑，輕輕搖動PVDF膜，觀察顯影情況，當能夠清晰的看到顯色帶時，用蒸餾水在30分鐘內(nèi)分三次清洗PVDF膜以終止顯色反應(yīng)的繼續(xù)進行。
【實驗結(jié)果】
檢查膜上顯色結(jié)果，藍紫色帶所對應(yīng)的即是目標蛋白的位置。
                     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.

實驗材料】

1. 實驗器材

SDS/PAGE實驗相關(guān)材料；電轉(zhuǎn)移裝置；供電設(shè)備；PVDF膜（Millipore Immobion-P #IPVH 000 10）；Whatman 3MM 紙；其他工具：鑷子、海綿墊、剪子、手套、小塑料或玻璃容器、淺盤。

⑴ 10x轉(zhuǎn)移緩沖溶液（1L）：30.3g Trizma base（0.25M）, 144 g甘氨酸（1.92M）,加蒸餾水至1L, 此時pH約為8.3，不必調(diào)整。

⑵ 1x轉(zhuǎn)移緩沖溶液（2L）：在1.4L蒸餾水中加入400 ml甲醇及200 ml10x 轉(zhuǎn)移緩沖溶液。

⑶ TBS 緩沖溶液:將1.22g Tris （10 mM）和8.78g NaCl（150 mM）加入到1L蒸餾水中，用HCl調(diào)節(jié)pH至7.5。

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

⑸ 一抗：兔抗待測蛋白抗體（多克隆抗體）。

（6） 二抗：辣根過氧化物酶標記羊抗兔。

⑺ 3% 封阻緩沖溶液（0.5L）：牛血清白蛋白15mg加入TBS緩沖溶液并定容至0.5L，過濾，在4°C 保存以防止細菌污染。

⑻ 0.5%封阻緩沖溶液（0.5L）：牛血清白蛋白2.5mg加入TTBS緩沖溶液并定容至0.5L，過濾，在4°C 保存以防止細菌污染。

⑼ 顯影試劑：1ml 氯萘溶液 （30mg/ml甲醇配置），加入10 ml甲醇，加入TBS緩沖溶液至50 ml，加入30 ul 30% H₂O₂。

⑽ 染色液：1g氨基黑18B （0.1%），250ml異丙醇（25%）及100ml乙酸（10%）用蒸餾水定容至1L。

⑾ 脫色液：將350ml異丙醇（35%）和20 ml乙酸（2%）用蒸餾水定容至1L。

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