![]() Today more researchers are taking advantage of fluorescently conjugated secondary antibodies that eliminate the need for substrates and provides the ability to detect multiple proteins at the same time. If using an enzyme-based system, a few different substrates can be used, which produce either a colorimetric or chemiluminescence signal.Ĭhemiluminescence detection is the method of choice in many protein laboratories, as it provides high sensitivity and convenience for detection with a film or digital imaging equipment. The secondary antibody is typically tagged with a reporter enzyme like Horse Radish Peroxidase or a fluorophore.Īs before, any unbound secondary antibody is washed away. In this step, the membrane is incubated with the primary antibody specific to the target to allow the antibody to bind its target.Īny unbound primary antibody is washed away.Ī secondary antibody that is specific to the primary antibody is then added. Recent advancements in blocking agents have helped make this step faster and more efficient. Proteins in this blocking solution occupy the empty spaces on the membrane to help prevent detection antibodies in the next step from sticking to the membrane. To do this the membrane should first be incubated with a protein containing solution such as nonfat dry milk or a purified protein like bovine serum albumin, or BSA. This is a multi-step process, and the first step is minimizing nonspecific binding by using a blocking agent. Once complete, the efficiency of the transfer can be confirmed by staining the membrane with a reversible stain. However, new technologies have been developed that can cut the transfer time down to as little as 7 minutes. Using a traditional apparatus, the protein transfer process can take 30 to 60 minutes to complete. Once the proteins have been separated in the gel, they must be transferred to a solid support membrane, which facilitates protein detection through the use of antibodies specific to the target protein. In this video, we will cover the 3 major steps in generating a western blot: Separate, Transfer, and Detect.Īfter protein samples have been prepared, the mixture is separated by molecular weight using protein gel electrophoresis, which is a standard laboratory technique by which charged protein molecules are transported through a matrix by an electrical field. Western Blotting is the most widely used technique for protein detection.
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