蛋白質(zhì)印跡分析-WesternBlotanalysis

1、實(shí)驗(yàn)四 蛋白質(zhì)印跡分析【實(shí)驗(yàn)?zāi)康摹苛私獾鞍踪|(zhì)印跡法的基本原理及其操作和應(yīng)用?！緦?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(xiàng)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(jìn)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(diǎn)等采用不同的電泳方法進(jìn)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，

2、如牛血清白蛋白對膜進(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 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 分鐘延長到過 夜進(jìn)行。由于濕法的使用彈性更大并且沒有明顯浪費(fèi)更多的時(shí)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(biāo)記了特定的試劑， 如辣根

3、過氧化物酶。 這種標(biāo)記是利用辣根過氧化物酶所催化 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 對二抗的識別而識別一抗， 進(jìn)而判斷出目標(biāo)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(biāo)記系統(tǒng)?！緦?shí)驗(yàn)材料】1. 實(shí)驗(yàn)器材SDS/PAGE實(shí)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 ); 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。2. 實(shí)驗(yàn)試劑 10x 轉(zhuǎn)移緩沖溶液(1L)： 30.3g Trizma base(0.25

4、M), 144 g 甘氨酸(1.92M),加蒸餾水至 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 NaCI(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：兔抗待測蛋白抗體(多克隆抗體) 。(6) 二抗：辣根過氧化物酶標(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

5、溶液并定容至 0.5L , 過濾，在 4C 保存以防止細(xì)菌污染。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4C保存以防止細(xì)菌污染。 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% H 2O2。 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。(11)脫色液：將350ml異丙醇(35%)和20 ml乙酸(2%)用蒸餾水定容至1L。封閉和清洗加入二抗L底物檢測f底物蛋白質(zhì)印跡法基本操

6、作過程【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。夾心放置順序黑色篩孔板 海綿墊3MM PVDF 凝膠3MM 紙海綿墊 白色篩孔板制作膠膜夾心在一淺盤中打開轉(zhuǎn)移盒，將一個(gè)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

7、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(shí)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(shí)防止出現(xiàn)氣泡。電轉(zhuǎn)移連接電源，在4C條件下維持恒壓100v，1小時(shí)。3免疫檢測膜染色斷開電源，將轉(zhuǎn)移盒從轉(zhuǎn)移槽中移出，將轉(zhuǎn)移盒的各個(gè)部分分開。用鑷子將PVDF膜小心放入一個(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的 膜放入另一個(gè)

8、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。膜的封閉和清洗對于沒有進(jìn)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(dòng)至少1小時(shí)。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。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(dòng)1小時(shí)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。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(dòng)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 1

9、0分鐘。檢測倒掉TTBS緩沖溶液，并加入顯影劑，輕輕搖動(dòng)PVDF膜，觀察顯影情況，當(dāng)能夠清晰的看到顯色帶時(shí)，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(jìn)行?！緦?shí)驗(yàn)結(jié)果】檢查膜上顯色結(jié)果，藍(lán)紫色帶所對應(yīng)的即是目標(biāo)蛋白的位置?！舅伎碱}】1. 蛋白質(zhì)印跡法的特點(diǎn)是什么？2. 請解釋什么是BSA ?并說明它在本實(shí)驗(yàn)中的作用。3請說明二抗在蛋白質(zhì)印跡法中的生物學(xué)功能。4.如何保存抗體？Experiment 16 Western Blot Analysis【 Purpose】Comprehend the theory of Western blotting; understand it

10、s 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 pol

11、yclonal 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 (isoe

12、lectric 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 s

13、ite 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

14、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 is submerged in transfer buffer for electrophoresis, which may take as l

15、ittle 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. Typi

16、cally, 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 recog

17、nize first antibody through recognizing second antibody, and then identify the position of target protein. Other detection systems include alkaline phosphatase and 125I labels. 【 Materials 】1 Apparatus:Apparatus of SDS-PAGE, Electroblotting Apparatus, Power supply, PVDF membrane (Millipore Immobion-

18、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 Glycine (1.92 M), pH shouldbe 8.3; without adjustment. 1x transfer buffer (2 L): 400 ml

19、 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 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-

20、rabbit-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 ba

21、cterial 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 fina

22、l volume 1L.(11) Destaining buffer: Add 350ml isopropanol (35%) and 2 ml acetic acid(2%) to distilled water with final volume 1L. ElectrophoresisMembra ne tran sferAddition of.EPrimary an tibodyBlock ing andWash ingAddition ofThe basic procedure of Wester n blott ing【Procedure 】1. . Separation of Pr

23、oteinRun an electrophoretic separation of known antigenic proteins. The method 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

24、 right-hand corner of gel for orientation and put gel in transfer buffer until 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 ofgel. Incubate in methanol for about 1 min on a rocker at room temp. R

25、emove methanol and equilibrate membra ne in 1x tran sfer buffer un til ready 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

26、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 sec ond well-soaked pad. Close the san dwich with the white piece of the ca

27、ssette. Mount the san dwich in the tran sfer tank; put the black sides n ear the black side of the device. Fill the buffer tank with the tran sfer buffer.Cassette (black piece) Sponge pad 3MM paperPVDF membrane gel3MM paperSponge padCassette (White piece)The arra ngeme nt of san dwich Electrotransfe

28、r:Attach the electrodes. Set the power supply to 100V (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 fo

29、r 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 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 , rock gently for at least 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 ap