Western Blot流程(自CST網(wǎng)站及《精編分子生物學(xué)實(shí)驗(yàn)指南》).

1、 HYPERLINK l 書簽4 根本原理、分類流程： HYPERLINK l 書簽1 自CST網(wǎng)站流程： HYPERLINK l 書簽2 自?精編分子生物學(xué)實(shí)驗(yàn)指南?Western Blot是將蛋白質(zhì)轉(zhuǎn)移到膜上，然后利用抗體進(jìn)行檢測。對表達(dá)蛋白，可用相應(yīng)抗體進(jìn)行檢測。對新基因的表達(dá)產(chǎn)物，可通過融合局部的抗體檢測Western Blot中文一般稱為蛋白質(zhì)印跡。它是分子生物學(xué)、生物化學(xué)和免疫遺傳學(xué)中常用的一種實(shí)驗(yàn)方法。其根本原理是通過特異性抗體對凝膠電泳處理過的細(xì)胞或生物組織樣品進(jìn)行著色。通過分析著色的位置和著色深度獲得特定蛋白質(zhì)在所分析的細(xì)胞或組織中的表達(dá)情況的信息。蛋白質(zhì)印跡的創(chuàng)造者一般認(rèn)為

2、是美國斯坦福大學(xué)的喬治斯塔克George Stark。在尼爾伯奈特Neal Burnette于1981年所著的?分析生物化學(xué)?Analytical Biochemistry中首次被稱為Western Blot。 原理Western Blot與Southern印跡雜交或Northern雜交方法類似，但Western Blot采用的是聚丙烯酰胺凝膠電泳，被檢測物是蛋白質(zhì)，“探針是抗體，“顯色用標(biāo)記的二抗。經(jīng)過PAGE別離的蛋白質(zhì)樣品，轉(zhuǎn)移到固相載體例如硝酸纖維素膜NC膜上，固相載體以非共價(jià)鍵形式吸附蛋白質(zhì)，且能保持電泳別離的多肽類型及其生物學(xué)活性不變。以固相載體上的蛋白質(zhì)或多肽作為抗原，與對應(yīng)的抗

3、體起免疫反響，再與酶或同位素標(biāo)記的第二抗體起反響，經(jīng)過底物顯色或放射自顯影以檢測電泳別離的特異性目的基因表達(dá)的蛋白成分。該技術(shù)也廣泛應(yīng)用于檢測蛋白水平的表達(dá)。 分類Western Blot顯色的方法主要有以下幾種：i. 放射自顯影ii. 底物化學(xué)發(fā)光ECLiii. 底物熒光ECFiv. 底物DAB呈色現(xiàn)常用的有底物化學(xué)發(fā)光ECL和底物DAB呈色，體同水平和實(shí)驗(yàn)條件的是用第一種方法，目前發(fā)表文章通常是用底物化學(xué)發(fā)光ECL。只要買現(xiàn)成的試劑盒就行，操作也比擬簡單，原理如下二抗用HRP標(biāo)記：反響底物為過氧化物+魯米諾，如遇到HRP，即發(fā)光，可使膠片曝光，就可洗出條帶。編輯本段其他值得一提的是，wes

4、tern blot 這個(gè)名稱的由來很有意思。最開始做印跡工作的是一個(gè)叫做Southern的科學(xué)家，但印跡的對象是DNA鏈，他把這種技術(shù)稱為Southern blot，后來類似的出現(xiàn)了兩個(gè)過程相似，但是對象不同的印跡方法，一個(gè)針對RNA，一個(gè)對蛋白質(zhì)，人們分別把這兩種技術(shù)的稱為Northern和Western，與這兩個(gè)技術(shù)的創(chuàng)造人沒有關(guān)系了。 HYPERLINK l 書簽3 返回 Western Immunoblotting Protocol (Primary Ab Incubation In BSA) For Western blots, incubate membrane with dilu

5、ted antibody in 5% w/v BSA, 1X TBS, 0.1% Tween-20 at 4C with gentle shaking, overnight. HYPERLINK l 書簽3 返回Products available from Cell Signaling Technology are linked by their respective catalog numbers.A. Solutions and ReagentsNOTE: Prepare solutions with Milli-Q or equivalently purified water.1X P

6、hosphate Buffered Saline (PBS). 1X SDS Sample Buffer: ( HYPERLINK :/ cellsignal /products/7722.html #7722, HYPERLINK :/ cellsignal /products/7723.html #7723) 62.5 mM Tris-HCl (pH 6.8 at 25C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue or phenol red. Transfer Buffer: 25 mM Tris b

7、ase, 0.2 M glycine, 20% methanol (pH 8.5). 10X Tris Buffered Saline (TBS): ( HYPERLINK :/ cellsignal /products/9997.html #9997) To prepare 1 liter of 10X TBS: 24.2 g Tris base, 80 g NaCl; adjust pH to 7.6 with HCl (use at 1X). Nonfat Dry Milk: ( HYPERLINK :/ cellsignal /products/9999.html #9999) (we

8、ight to volume w/v). Blocking Buffer: 1X TBS, 0.1% Tween-20 with 5% w/v nonfat dry milk; for 150 ml, add 15 ml 10X TBS to 135 ml water, mix. Add 7.5 g nonfat dry milk and mix well. While stirring, add 0.15 ml Tween-20 (100%). Wash Buffer: 1X TBS, 0.1% Tween-20 (TBS/T). Bovine Serum Albumin (BSA): (

9、HYPERLINK :/ cellsignal /products/9998.html #9998). Primary Antibody Dilution Buffer: 1X TBS, 0.1% Tween-20 with 5% BSA; for 20 ml, add 2 ml 10X TBS to 18 ml water, mix. Add 1.0 g BSA and mix well. While stirring, add 20 l Tween-20 (100%). Phototope-HRP Western Blot Detection System: ( HYPERLINK :/

10、cellsignal /products/7071.html #7071 anti-rabbit) or ( HYPERLINK :/ cellsignal /products/7072.html #7072 anti-mouse) Includes biotinylated protein ladder, secondary ( HYPERLINK :/ cellsignal /products/7074.html #7074 anti-rabbit) or ( HYPERLINK :/ cellsignal /products/7076.html #7076 anti-mouse) ant

11、ibody conjugated to horseradish peroxidase (HRP), anti-biotin antibody conjugated to HRP, LumiGLO chemiluminescent reagent and peroxide. Prestained Protein Marker, Broad Range (Premixed Format): ( HYPERLINK :/ cellsignal /products/7720.html #7720). Biotinylated Protein Ladder Detection Pack: ( HYPER

12、LINK :/ cellsignal /products/7727.html #7727). Blotting Membrane: This protocol has been optimized for nitrocellulose membranes, which CST recommends. PVDF membranes may also be used. B. Protein BlottingA general protocol for sample preparation is described below.Treat cells by adding fresh media co

13、ntaining regulator for desired time. Aspirate media from cultures; wash cells with 1X PBS; aspirate. Lyse cells by adding 1X SDS sample buffer (100 l per well of 6-well plate or 500 l per plate of 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microce

14、ntrifuge tube. Keep on ice. Sonicate for 1015 seconds for complete cell lysis and to shear DNA (to reduce sample viscosity). Heat a 20 l sample to 95100C for 5 minutes; cool on ice. Microcentrifuge for 5 minutes. Load 20 l onto SDSgel (10 cm x 10 cm). NOTE: CST recommends loading prestained molecula

15、r weight markers ( HYPERLINK :/ cellsignal /products/7720.html #7720, 10 l/lane) to verify electrotransfer and biotinylated protein ladder ( HYPERLINK :/ cellsignal /products/7727.html #7727, 10 l/lane) to determine molecular weights. Electrotransfer to nitrocellulose or PVDF membrane. C. Membrane B

16、locking and Antibody IncubationsNOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.(Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 minutes at room temperature. Incubate membrane in 25 ml of blocking buffer

17、 for 1 hour at room temperature. Wash three times for 5 minutes each with 15 ml of TBS/T. Incubate membrane and primary antibody (at the appropriate dilution) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4C. Wash three times for 5 minutes each with 15 ml of TBS/T. I.

18、For Unconjugated Primary AntibodiesIncubate membrane with appropriate HRP-conjugated secondary antibody (1:2000) and HRP-conjugated anti-biotin antibody (1:1000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hour at room temperature. Wash three times

19、for 5 minutes each with 15 ml of TBS/T. II. For HRP Conjugated Primary AntibodiesSkip to Detection of Proteins (Step D).III. For Biotinylated Primary AntibodiesIncubate membrane with HRP-Streptavidin (at the appropriate dilution) in milk for one hour with gentle agitation at room temperature. Wash three times for 5 minutes each with 15 ml of TBS/T. D. Detection of ProteinsIncubate membrane with 10 ml LumiGLO (0.5 ml