Question

What type of assay allows you to identify proteins in a complex?

Answer 1

Western blot is a assay used to detect proteins from a complex.

Western blot is used to separate and identify proteins. In this technique a mixture of proteins is separated based on their molecular weight, and their activity , through gel electrophoresis. The membrane is then incubated with labels antibodies specific to the protein of wanted.By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three steps,  separation by size, transfer to a solid support, and marking target protein using a proper primary and secondary antibody to visualize. Western blot is used in research to separate and identify proteins from a mixture. In this technique a mixture of proteins is separated based on molecular weight, and thus by type of through gel electrophoresis. These results are then transferred to a membrane producing a band for each protein. The membrane is then incubated with labels antibodies specific to the protein of interest.The unbound antibody is washed off leaving only the bound antibody to the protein of interest. The bound antibodies are then detected by developing the film. As the antibodies only bind to the protein of wanted only one band should be visible. The thickness of the band corresponds to the amount of protein present , and  indicate the amount of protein is present.

Cell suspension  are the most common form of sample used for western blot. Protein extraction attempts to collect all the proteins in the cell cytosol. This procedure should be done in a cold temperature with protease inhibitors to prevent denaturing of that  proteins. After extracting the protein, Protein concentration is often measured using a spectrophotometer. Using this concentration allows to measure the mass of the protein that is being loaded into each well. After determine the volume of the sample, it is diluted into a loading buffer, which After determining the appropriate volume,  which contains glycerol so that the samples sink easily into the wells of the gel. A tracking dye (bromophenol blue) is also present in the buffer allowing the researcher to find the separation has progressed. The sample is heated after being diluted into a loading buffer, in order to denature the higher order structure.  Denaturing the high structure gives that the negative charge of amino acids is not neutralized, enabling the protein to move in an electric field (applied during electric field). In this technique to detect the positive and negative controls for the sample. For a positive control a known source of target protein, such as purified protein or a control lysate is used. This helps to confirm the identity of the protein, and the activity of the antibody. A negative control is a null cell line, is used as well to confirm that the staining is not nonspecific. proteins in a sample of tissue of extract. In simple steps, it include the major steps;

(1) buffers preparation, (2) samples preparation, (3) gel electrophoresis, (4) protein transfer, (5) membrane blocking, (6) antibody incubation, (7) detection and imaging, (8) Data analysis.

In simple method, the sample undergoes protein denaturation, followed by gel electrophoresis. The primary antibody is created that recognises and binds to a specific target protein. The electrophoresis membrane is washed in a solution containing the primary antibody, before excess antibody is washed off. A secondary antibody is added which recognises and binds to the primary antibody. The secondary antibody is visualised through various methods such as staining, immunofluorescence, and radioactivity, allowing indirect detection of the specific target protein.