western-blot

研究人员发明了一种能同时检测大量蛋白的新方法，这种新方法效果与传统的Western Blotting一样好，但是时间可以大大缩短，比传统快48倍。

免疫印迹（immunoblotting）又称蛋白质印迹（western blot），它是根据抗原抗体的特异性结合检测复杂样品中的某种蛋白的方法。

SDS 平板胶片铸造︰1） 整理出两组玻璃片及白板铸胶组合，先用酒精拭净，并选择合适的间隔条（0.75 mm） 组合起来。请熟悉铸胶三明治组合的正确组装方式，以免灌入的胶液漏出来。2） 三明治组合后直立站好，准备所要浓度的SDS 胶体溶液如表4.2. 两片0.75mm 厚的平板胶片约需10 mL 分离胶体溶液，以及5 mL 焦集胶体溶液。APS液到最后才加入，未加APS 以前可在真空中抽去溶液中的气体……

蛋白质经SDS-PAGE后，胶片浸入转印缓冲液，蛋白质可被转印到硝化纤维纸（nitrocellulose） 上，先经尿素洗去SDS,并使蛋白质回复原态抗原性，可使用抗体进行免疫染色 （Towbin et al, 1979）。

Western-Blotting方法：膜的准备：将PVDF膜切成条浸在甲醇中，室温条件下在摇床上摇1min,去除甲醇后加入1×TBST备用。B. 膜转印：1. 细胞或组织裂解液进行SDS-PAGE电泳2. 用夹心法电转至PVDF膜3. 海绵和滤纸浸泡在转印缓冲液预湿润……

Western Blot 印迹法是把通过电泳分离的蛋白质组分从凝胶转移至一种固相支持物，通过抗体与附着于固相支持物的靶蛋白的抗原表位发生特异性反应进行检测。Western Blot 既可以定性，又可以半定量，是初步鉴定靶蛋白表达的最方便也是最通用的方法。

1. Resolve sample proteins and controls via polyacrylamide gel electrophoresis. Transfer proteins to nitrocellulose using standard methods. 2. Remove the blot from the transfer apparatus and soak in T ...

1. 溶液准备： 转印缓冲液：0.025M Tris base ， 0.192 M甘氨酸 ， 30%甲醇 10×TBST：250mM Tris-HCl （ pH 8.0 ） ， 1.25M NaCl ， 0.5%Tween20 封闭液：1×TBST ， 3%脱脂奶粉 洗涤液：1×TBST 2. 实验程序： A. 膜的准备： 将PVDF膜切成条浸在甲醇中，室温条件 ...

METHOD for Western Blots: 1.While your SDS-PAGE gel is runningmake your transfer buffer and chill to 4℃.Check that you have a frozen buffer dam is ready (stored in freezer next to Shikhatop shelf to t ...

DunnAnal.Biochem.1986: 157 GeorgiaTimes"1.5L GeorgiaTimes"1.0L GeorgiaTimes"10mM NaHCO3 ...

ECL or autoradiography? ECL is an appealing technique because it is quick and very sensitive and does not expose the investigator to radioactivity.The use of radioiodinated protein A to detect bound a ...

The following tips can be used to overcome the most common problems encountered during Western blotting. Smeared Pattern or Distorted Bands Uneven contact between gel and membrane: cassettes used sh ...

Materials Blot Cell BA 83 0.2 µm pore nitrocellulose sheets Buffer PBS-Tween 20 Antigenic proteins antibodies and horseradish peroxidase labeled antiglobulins Procedure Run an electrophore ...

Western Blot 1、Run protein gel. Set up blot: 2、Prepare two tupperware containers one with dH2Oand one with 1/2X Transfer Buffer (TB). 3、Remove one glass plate from the gel by twisting a spacer betwee ...

REAGENTS ECL Western blotting kit (Amersham Life Science; cat# RPN2108): contains second antibodies for both mouse and rabbit substrate and milk blocker (the milk blocker is not normally used when us ...

Western Blotting with Alkaline Phosphatase Conjugates SAMPLE PREPARATION For Protein Concentration Determination of Cell Culture 1.Decant medium from 10cm dish of adherent cells and rinse plate rapidl ...

Western Blotting with Biotinylated Antibodies SAMPLE PREPARATION For Protein Concentration Determination of Cell Culture 1.Decant medium from 10cm dish of adherent cells and rinse plate rapidly with p ...

SAMPLE PREPARATION For Protein Concentration Determination of Cell Culture 1.Decant medium from 10cm dish of adherent cells and rinse plate rapidly with phosphate-buffered saline (PBS). 2.Aspirate ex ...

Sample Preparation For Protein Concentration Determination of Cell Culture 1.Decant medium from 10cm dish of adherent cells and rinse plate rapidly with phosphate-buffered saline (PBS). 2.Aspirate e ...

Running Protein Gels Solutions 10X Running Buffer (0.25 M Tris 1.92 M glycine 1% SDS) 121 g Tris 577 g glycine 40 g SDS ddh20 to 4 L (check pH at 1:10 dilution (pH=~8.8)). 5X Sample Buffer (0.3125 M T ...