In WB polyacrylamide gels are used for protein separation and the method is therefore called polyacrylamide gel electrophoresis (PAGE) when using native condition. An electric field is applied over the gel that causes the charged molecules to move. Gel electrophoresisĪfter sample preparation the extract is ready to be loaded to separate the proteins according to size by gel electrophoresis. Preferably positive and negative controls are included in the set up to confirm identity of the protein as well as the activity of the antibody. Heat is applied on the samples in order to break the structures of the protein, which will help keeping the negative charge from neutralization ( Mahmood & Yang, 2012). The glycerol is used to simplify the loading by raising the density of the extract and the dye is added to visualize the sample. The extract is then diluted with loading buffer consisting of glycerol and a dye (e.g. Usually a biochemical assay is used in order to determine the protein concentration. It is important to determine the total protein concentration of the generated extract to be able to load a specific amount on the gel to enable comparison between samples. Different types of filtration and centrifugation methods are applied to further prepare the samples. Buffers are added to lyse the cells and solubilize the proteins and often an inhibitor is added to prevent denaturation or degradation. Usually the tissue needs to be broken down by blending, homogenization, or sonication. tissue, cells, or other solution, which is going to be analyzed. The first step of a WB is to prepare the sample, e.g. However, WB is a very common method and almost all available commercial antibodies have been validated using this method. The result form the WB is not always easy to interpret as the size of the protein may vary from the theoretical weight due to posttranslational modifications, such as glycosylation, or interactions with other proteins. Moreover, the specificity of the binding to the target and a low cross reactivity are important features as well. The outcome of a WB experiment depends on three important factors the ability of the antibody to bind a specific protein, the strength of the interaction, and the concentration of the protein of interest itself. In addition, the non-linear relation of the generated signal across the concentration range of the samples is also an aspect of consideration when interpreting the results. When analyzing the results, variations between lanes regarding loading and transfer rates between blots, must be taken into consideration. The setup of the experiment can be varied in many ways to best suit the specific inquiry. The separation on the gel is not only due to size but also to some extent depending on the molecular charge, hydrophobic regions, and degree of denaturation. An image is taken of the membrane and the result is analyzed.īy adding a separate marker solution to one of the wells in the gel, it is possible to estimate the size of the protein in addition to the antibody interactions that are used to verify the specific protein. To visualize the protein of interest the membrane is commonly first probed using a primary protein-specific antibody followed by a labeled secondary antibody used for detection. After the transfer, the membrane is blocked in order to prevent unwanted membrane-protein interaction in the following steps. In order to further analyze the proteins, they are transferred onto a membrane in a procedure called blotting. Samples are prepared and loaded on to a gel and during the electrophoresis the negatively charged proteins move toward the positively charged anode. The setup consists of a standard set of seven steps, Figure 1.įigure 1. Towbin et al described electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets where the original gel pattern was accurately obtained. Towbin et al in 1979 ( Towbin, Staehelin, & Gordon, 1979) and two years later given its name by W. It is built on a technique that involves transferring, also known as blotting, proteins separated by electrophoresis from the gel to a membrane where they can be visualized specifically. Western Blot (WB) is a common method to detect and analyze proteins.

Western blot protein size ladder