when working with proteins one cannot get around the technique called protein immunoblot or simply the western blot western blotting is a method to confirm the presence of a specific target protein in a sample before the analytical technique western blood comes into play everything starts with an sds page upon treatment with sds all proteins in a sample are denatured and covered by negative charges the proteins have a similar master charge ratio and travel through the gel to the positively charged anode this allows to separate proteins according to their molecular weight after staining the polyacrylamide gel protein bands become visible marker proteins as a reference help to determine the molecular weight of proteins in the sample this band here for example contains proteins with a molecular weight of approximately 60 kilodalton however that will not reveal which proteins are present in this sample it could be the protein of interest which has a molecular weight in this range but the band could have also been caused by other proteins to confirm the presence of a specific protein a western blood has to be performed the detection of the protein of interest is based on antibodies designed to exclusively bind epitopes of the target protein the western blot setup starts with the gel containing the separated proteins this is placed on top of a membrane often in nitrocellulose membrane this membrane is capable to bind large amounts of protein later the idea is to transfer the proteins from the inside of the gel onto the membrane this gel membrane construct is flanked on both sides by filter papers all parts here are soaked in buffer to facilitate the electricity transmission later finally everything is fixed between the cathode and the anode the electric current pulls the negatively charged proteins which are located in the gel to the positively charged a node this transfers the proteins onto the membrane from now on the membrane has to be incubated several times the first step includes blocking therefore the membrane is incubated with milk or bsa blocking should prevent antibodies from unspecifically binding to the membrane or to other proteins after blocking the membrane can be incubated with the primary antibody this antibody should be selected to only bind to the protein of interest after incubation with this primary antibody a washing step will wash away the unbound antibodies while not removing the antibodies that are bound to the protein of interest the next step involves the incubation with a secondary antibody which has to be specific for the primary antibody after washing only those secondary antibodies remain which are bound to the primary antibody the detection of the protein of interest is the last step in western blood analysis for that purpose the secondary antibodies are conjugated to an enzyme for many western blood assays secondary antibodies are conjugated with horseradish peroxidase the protein of interest is bound by a primary antibody the secondary antibody binds to the primary antibody and when the membrane is incubated in a substrate mixture the enzyme converts the substrate thereby producing a chemiluminescent signal that visualizes the protein band the presence of the target protein in these samples has been confirmed make sure to subscribe to stay updated for new videos and please like this video if it was helpful to you thanks