Western blotThe Western Blot is a biotechnology technique enabling the detection, quantification and relative mass evaluation. It consists in a gel electrophoresis followed by a labeling step with antibodies detecting the protein.
Gel electrophoresisGel electrophoresis separates proteins given their molecular weight, isoelectric point or charges. In SDS-PAGE electrophoresis, the protein is denatured and negatively charge thanks to sodium dodecyl sulfate (SDS). It can migrate through the polyacrylamide gel toward the positive anode. The bigger the protein, the slower it migrates. Therefore, the proteins can be separated based on their molecular weight. The acrylamide concentration in the gel can be optimized to adapt the resolution of the separation.
TransferThe separated proteins are transferred on a solid membrane. The membrane may be in nitrocellulose for example. A common technique to achieve this transfer is electro-blotting. Here again, proteins are attracted on the membrane thanks to an electric current. All transferred proteins may then be stained to evaluate the transfer efficiency. The membrane is then flooded with a solution containing proteins that are not of interest. This blocking step avoids the non-specific bounds between the membrane and the detection antibodies.
LabelingOnce the membrane is ready, the protein of interest is targeted by primary antibodies specifically. The antibodies solution is then removed and the unbound antibodies washed away. Secondary antibodies are then bound to the primary antibodies and washed in the same manner to avoid noise in the detection. The secondary antibodies used in this process are bound to enzymes that can emit signal used to detect the targeted protein. The signal and thus detection may be of different types from colorimetric to fluorescent or chemiluminescent, or even radioactive detection. The thickness of the band and the intensity of the signal may be analyzed to evaluate the quantity of the protein.