western-blot英文版讲稿.pptVIP

Western Blotting Introduction The method originated in the laboratory of George Stark at Stanford. In 1981，for the first time in called Western Blot was in the Analytical Biochemistry by Neal Burnette. Western blot is a technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies. So called since it has some similarity to a Southern blot. We can use this technique to identify a target protein in a complex mixture, and we can also use it to measure it’s expression level. Protein is tested object,antibody is the probe，secondary antibody is the colour reagents. Advantage high resolution of electrophoretic technique Specific and sensitive immunoreaction Target protein:1-5ng The Flow Path Tissue preparation ↓ SDS-PACE ↓ Transfer ↓ Blocking ↓ Detect antibody ↓ Reveral protein of interest Tissue preparation Samples can be taken from whole tissue or from cell culture. Solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. SDS-PACE Poly Acrylamide Gel Electrophoresis（PAGE）is the best method in protein separate。 It uses gel electrophoresis to separate native proteins by 3-D structure or dena-tured proteins by the length of the polype-ptide. SDS-PACE SDS-PACE Transfer In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The primary method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. Wet transfer Semidry transfer PonceauS red Coomassie Brilliant Blue gel membrane PVDF/NC Blocking Since the membrane has been chosen for its ability to bind protein and as both antibodies and the target are proteins, steps must be taken to prevent the interactions between the membrane and the