Western-Blot過(guò)程步驟詳解中英文對(duì)照

1、精品文檔實(shí)驗(yàn)四 蛋白質(zhì)印跡分析【實(shí)驗(yàn)?zāi)康摹?了解蛋白質(zhì)印跡法的基本原理及其操作和應(yīng)用。 【實(shí)驗(yàn)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(cè)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(cè)蛋白既可以是粗提物也可以經(jīng)過(guò)一定的分離和純化, 另外這項(xiàng)技術(shù)的應(yīng)用需要利用待測(cè)蛋白的單克隆或多克隆抗體進(jìn)行識(shí)別。如圖所示，可溶性抗原，也就是待測(cè)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(diǎn)等采用不同的電泳方法進(jìn)行分離；通過(guò)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

2、白蛋白對(duì)膜進(jìn)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(gè)過(guò)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(shí)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(shí)間可從45 分鐘延長(zhǎng)到過(guò)夜進(jìn)行。由于濕法的使用彈性更大并且沒(méi)有明顯浪費(fèi)更多的時(shí)間和原料，因此我們?cè)谶@里只描述濕法的基本操作過(guò)程。對(duì)于目的蛋白的識(shí)別需要采用能夠識(shí)別一抗的第二抗體。該抗體往往是購(gòu)買(mǎi)的成品，已經(jīng)被結(jié)合或標(biāo)記了特定的試劑，如辣根過(guò)氧化物酶。這種標(biāo)記是利用辣根

3、過(guò)氧化物酶所催化的一個(gè)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(guò)對(duì)二抗的識(shí)別而識(shí)別一抗， 進(jìn)而判斷出目標(biāo)蛋白所在的位置。其他的識(shí)別系統(tǒng)包括堿性磷酸125酶系統(tǒng)和I 標(biāo)記系統(tǒng)?！緦?shí)驗(yàn)材料】1. 實(shí)驗(yàn)器材SDS/PAG改驗(yàn)相關(guān)材料；電轉(zhuǎn)移裝置；供電設(shè)備；PVDF膜（Millipore Immobion-P #IPVH000 10）; Whatman3MM紙；其他工具：鐐子、海綿墊、剪子、手套、小塑料或玻璃容器、淺盤(pán)。2. 實(shí)驗(yàn)試劑 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

4、至1L, 此時(shí) pH 約為8.3，不必調(diào)整。 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。 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。 TTBS buffer ：在 1L TBS 緩沖溶液中加入0.5ml Tween 20 （ 0.05%） 。 一抗：兔抗待測(cè)蛋白抗體（多克隆抗體）。（6） 二抗：辣根過(guò)氧化物酶標(biāo)記羊抗兔。 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

5、.5L ， 保存以防止細(xì)菌污染。C 4 過(guò)濾，在， 0.5LTTBS 緩沖溶液并定容至0.5L） ：牛血清白蛋白2.5mg 加入 0.5%封阻緩沖溶液（保存以防止細(xì)菌污染。4 C 過(guò)濾， 在緩沖溶液TBS10 ml 甲醇， 加入 （30mg/ml 甲醇配置）， 加入 顯影試劑：1ml 氯萘溶液Oo至50 ml ,加入30 ul 30% H 22用蒸儲(chǔ)水定容乙酸 (10%)(25%)及100m1250ml染色液：1g氨基 黑18B (0.1%),異丙醇(10) 1L。至。2%)用蒸儲(chǔ)水定容至 1L異丙醇350m1(35%)和20 ml乙酸( 脫色液：將加入一抗膜轉(zhuǎn)移泳電E EEE加入二抗封閉和清

6、洗 底物物產(chǎn)EE 物產(chǎn)底 物 檢測(cè) 蛋白質(zhì)印跡法基本操作過(guò)程【實(shí)驗(yàn)操作】1.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(jìn)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(shí)驗(yàn)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(gè)小口以便定位，小心放入轉(zhuǎn)移緩沖溶液中待用。2.電轉(zhuǎn)移準(zhǔn)備PVDF膜分鐘，再根據(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 移至轉(zhuǎn)移緩沖溶液中待用。1泡.黑色篩孔板1 1海綿墊11| ., 3MM.1 . 1 PVDF11凝膠1 - - 1. 3MM 紙1 - 海綿墊11.白色篩孔板夾心放置順序制作膠膜夾心將一

7、個(gè)預(yù)先用轉(zhuǎn)移緩沖溶液浸泡過(guò)的海綿墊放在轉(zhuǎn)移盒的黑色在一淺盤(pán)中打開(kāi)轉(zhuǎn)移盒， 3MM3MM紙，小心地將膠板放在篩孔板上，在海綿墊的上方放置經(jīng)轉(zhuǎn)移緩沖溶液浸濕的膜放在膠的上方同時(shí)注意排除氣泡，再在膜的上方放上紙上，并注意排除氣泡。將PVDF關(guān)閉放置另一張浸泡過(guò)的海綿墊，3MM 一張同樣用轉(zhuǎn)移緩沖溶液浸濕過(guò)的紙并趕出氣泡，轉(zhuǎn)移盒的黑色篩 孔板貼近轉(zhuǎn)移槽的黑色端，將轉(zhuǎn)移盒按照正確的方向放入轉(zhuǎn)移槽中，轉(zhuǎn)移盒。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(shí)防止出現(xiàn)氣泡。電轉(zhuǎn)移1小時(shí)。4。連接電源，在C條件下維持恒壓100v,免疫檢測(cè)3.膜染色 膜斷開(kāi)電源，將轉(zhuǎn)移盒從轉(zhuǎn)移槽中移出，將轉(zhuǎn)移盒的各個(gè)部

8、分分開(kāi)。用鐐子將PVDF的小心放入一個(gè)干凈的容器中，用 TBS緩沖溶液進(jìn)行短暫清洗，從膜上剪下一條寬約5mm分分鐘，然后在脫色液中脫色130膜放入另一個(gè)干凈的容器中。將這條膜在染色液中浸泡膜上。，鐘確定蛋白質(zhì)已經(jīng)轉(zhuǎn)移到PVDF膜的封閉和清洗1 封閉緩沖溶液,輕輕搖動(dòng)至少,對(duì)于沒(méi)有進(jìn)行染色的膜，首先倒出TBS 緩沖溶液加入3% 53TBS緩沖溶液清洗次, 每次分鐘。3%小時(shí)。 倒掉封閉緩沖溶液，并用 一抗小時(shí)以上。1 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(dòng)TBS 倒掉 10分鐘。緩沖溶液清洗兩次，每次從容器中倒出一抗及封閉緩沖溶液，用TTBS 二抗分鐘，倒出TT

9、BS 緩沖溶液，加入5 ml 0.5%30 封閉緩沖溶液及適量的二抗。輕輕搖動(dòng)分鐘。TTBS 倒出二抗及封閉緩沖溶液，用緩沖溶液清洗兩次，每次10 檢測(cè)膜，觀察顯影情況，當(dāng)能夠清緩沖溶液，并加入顯影劑，輕輕搖動(dòng)PVDF 倒掉 TTBS晰的看到顯色帶時(shí)，用蒸餾水在膜以終止顯色反應(yīng)的繼續(xù)進(jìn)行。PVDF30 分鐘內(nèi)分三次清洗【實(shí)驗(yàn)結(jié)果】檢查膜上顯色結(jié)果，藍(lán)紫色帶所對(duì)應(yīng)的即是目標(biāo)蛋白的位置。L國(guó)考題】i.蛋白質(zhì)印跡法的特點(diǎn)是什么？2.請(qǐng)解釋什么是 BSA?并說(shuō)明它在本實(shí)驗(yàn)中的作用。3.請(qǐng)說(shuō)明二抗在蛋白質(zhì)印跡法中的生物學(xué)功能。?如何保存抗體4.Experiment 16 Western Blot Ana

10、lysis【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 c

11、rude 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 c

12、harge (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 sp

13、ecifically 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 c

14、alled electrophoretic blotting. The two common electrophoretic methods are: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 i

15、s 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

16、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 p

17、roduct that remains fixed to the membrane. Thus, it is possible to recognize first antibody through recognizing second antibody, and then identify 125I labels. the position of target protein. Other detection systems include alkaline phosphatase and學(xué)資學(xué)習(xí)網(wǎng)【 Materials 】1. Apparatus:Apparatus of SDS-PAGE

18、, 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.2. Reagents: 10x transfer buffer (1 L): 30.3 g Trizma base (0.25 M), 144 g Glycin

19、e (1.92 M), pH shouldbe 8.3; without adjustment. 1x transfer buffer (2 L): 400 ml Methanol, 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 andadjust pH to 7.5 with HCl. TTBS buffer: 1L TBS buffer add 0.5ml Tween 20 (0.05%).F

20、irst antibody: antibody against the target protein. (. horseradish peroxidase) Second antibody: goat anti-rabbit-HRPfinal to in TBS buffer Add 15mgBovine serum albumin 3% Blocking buffer (0.5 L):C to prevent bacterial contamination. volume 0.5 L, keep at 4 final to TBS buffer 2.5mg Bovineserum album

21、in in 0.5% Blocking buffer (0.5 L): AddC to prevent bacterial contamination.volume0.5 L, keep at 4 ml add 10 methanol), chlonoaphthol solution (30mg/ml in Developing reagent: 1mlO.methanol, add TBS buffer to 50 ml and add 30 ul 30% H 22ml and 100 250ml isopropanol (25%)amido Staining buffer: Add 1g

22、black 18B (0.1%),acetic acid (10%) to distilled water with final volume 1L.Destaining buffer: Add 350ml isopropanol(35%) and 2 ml acetic acid(2%) to distilled water (11)with final volume 1L.Addition of Electrophoresis Membrane transferPrimary antibodyE EEEAddition of Blocking andPrimary antibody Was

23、hingSubstrateProductEE ProductSubstrate DetectionThe basic procedure of Western blottingProcedure I. . Separation of ProteinRun an electrophoretic separation of known antigenic proteins. The method of separation decided by the characters of target protein, but for sufficiently transferring, the most

24、 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.2. . Electrotransfer Preparation of membraneCut a piece of PVDF membrane (Millipore Immobion-P

25、 #IPVH 000 10) according to the size of and methanol room temp. Remove min on a rocker at in gel. Incubate methanol for about 1 equilibrate membrane in 1x transfer buffer until ready to use.Arrange gel-membrane sandwich(2) In a shallow tray, open the transfer cassette. Put a well-soaked sponge pad o

26、n 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 and membrane the PVDF sheet of 3MM paper over remove any air bubbles. Place a wetted remov

27、e 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.Cassette (black piece) |Sponge pad |3MM paper

28、PVDF membrane gelI II .,3MM paper Sponge pad |Cassette (White piece) |,The arrangement of sandwichElectrotransfer: C.Attach the electrodes. Set the power supply to 100V (constant voltage) for 1h at 43.Immunodetection Membrane staining Disconnect transfer apparatus, remove transfer cassette, and peel

29、 3MM paper from membrane. Remove the membrane to a small container. Add 10 ml TBS buffer and wash for short time. Cut staining Stain another clean container. this stripe in in 5mm out one stripe with width and put been protein has to min min. buffer for 1 Destain for 30 in destaining buffer check wh

30、ether transferred from gel to membrane or not.Membrane blocking and washingrock gently for at For other part of membrane, pour off TBS buffer. Add 3% blocking buffer , least 1 h. Pour off 3% blocking buffer and rinse briefly with TBS buffer three times, 5 minutes forper time. First antibody Pour off TBS buffer.