The lower gel, called the separating, or resolving gel, is basic (pH 8.8), and has a higher polyacrylamide content, making the gel's pores narrower. The higher, stacking gel is slightly acidic (pH 6.8) and has a lower acrylamide concentration making a porous gel, which separates protein poorly but allows them to form thin, sharply defined bands. Western blot uses two different types of agarose gel: stacking and separating gel. A negative control is a null cell line, such as β-actin, is used as well to confirm that the staining is not nonspecific. This helps to confirm the identity of the protein, and the activity of the antibody. For a positive control a known source of target protein, such as purified protein or a control lysate is used. It is also very important to have positive and negative controls for the sample. Denaturing the high structure ensures that the negative charge of amino acids is not neutralized, enabling the protein to move in an electric field (applied during electrotransfer). The sample is heated after being diluted into a loading buffer, in order to denature the higher order structure, while retaining sulfide bridges. A tracking dye (bromophenol blue) is also present in the buffer allowing the researcher to see how far the separation has progressed. Using this concentration allows to measure the mass of the protein that is being loaded into each well by the relationship between concentration, mass, and volume.Īfter determining the appropriate volume of the sample, it is diluted into a loading buffer, which contains glycerol so that the samples sink easily into the wells of the gel. Protein concentration is often measured using a spectrophotometer. This eventually allows the researcher to ensure that the samples are being compared on an equivalent basis. Since tissue sample display a higher degree of structure, mechanical invention, such as homogenization, or sonication is needed to extract the proteins.Īfter extracting the protein, it is very important to have a good idea of the extract's concentration. This should be done in a cold temperature with protease inhibitors to prevent denaturing of the proteins. Protein extraction attempts to collect all the proteins in the cell cytosol. This will be followed by the theoretical explanation of the procedure, and in the later section, troubleshooting tips for common problems.Ĭell lysates are the most common form of sample used for western blot. The paper will first describe the protocol for western blot, accompanied by pictures to help the reader and theory to rationalize the protocol. The thickness of the band corresponds to the amount of protein present thus doing a standard can indicate the amount of protein present. As the antibodies only bind to the protein of interest, only one band should be visible. The bound antibodies are then detected by developing the film. The unbound antibody is washed off leaving only the bound antibody to the protein of interest.

The membrane is then incubated with labels antibodies specific to the protein of interest. These results are then transferred to a membrane producing a band for each protein. In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through gel electrophoresis. Western blot is often used in research to separate and identify proteins.

Human dot blot technique