WESTERN BLOTTING EXPLAINED скачать в хорошем качестве

WESTERN BLOTTING EXPLAINED

0:00-0:48 | What is Western Blotting? 0:48-1:58 | SDS-PAGE Step of Western Blotting 1:58-2:50 | Transfer Step of Western Blotting 2:50-3:38 | Antibody Detection Step of Western Blotting 3:38-4:20 | What is Western Blotting used for? Western blotting is used to detect a specific protein in a sample by combining gel electrophoresis and antibody recognition. In a nutshell, the sample’s proteins are first separated with the help of gel electrophoresis, then they are transferred out of the gel and onto a surface of a membrane. This membrane is then exposed to an antibody which has been selected for its quality to specifically bind to the target protein. In addition a radioactive or chemical tag has also been added to the antibody for detection. In the past on this channel, we have discussed both immunoassays and SDS page, and as you can see, western blotting, really you could say, combines these two techniques into one even greater technique. Let us take a closer look at this technique in action… 1. First the sample is separated using SDS PAGE, which separates the proteins according to their molecular weight. If you want to understand better how this technique works, I will link the video that focus solely on SDS PAGE by the end of this one! In a nutshell, due to the fact that the proteins contain a negative charge, and because of how the matrix has been constructed, once electricity is conducted through the gel, the proteins start moving from the negative to the positive end AND smaller proteins end up moving faster than larger ones. The end result looks like THIS, where the different bands corresponds to different sizes. However, another protein of a similar size might just be occupying the same space as the one you are interested. This is where the second part of Western blotting comes in handy! 2. To do this, the proteins are transferred into a PVDF membrane such that you can detect your protein of interest. This transfer process is done by putting the SDS-PAGE and the PVDF membrane next to each other surrounded by filter papers, in a transfer apparatus. This process, known as electroblotting facilitates movement of the proteins from the SDS-PAGE into the PVDF membrane. This is because an electrical current gets passed through the system and since, the proteins are negatively charged they move away from the gel and towards the membrane. This is why it is important to place the gel on the side of the cathode, which is negative, and the membrane on the side of the anode, which is positive! 3. Now, we can move to the antibody recognition part of western blotting. First however, the proteins have to be blocked by using milk or BSA, in order to prevent unspecific binding by the antibodies we will use to the membrane or other proteins. Then a primary antibody which has been selected to only bind to the protein of interest is added. Finally a secondary antibody which has been selected to only bind to the primary antibody is added. In addition, this secondary antibody also have been conjugated to an enzyme which when exposed to a specific substrate interacts with it, converting it to a different product. By observing where product has been created, we know where binding has occurred. So why is Western blotting useful? Well, the western blot is used for things like qualitative detection of singe proteins and protein-modifications. It is also used in the confirmatory HIV test and in the diagnosis of some forms of Lyme disease. It has also been used as a technique to detect doping in athletes.