Western Blots
Objectives
Protein Isolation: Protein isolation for a western blot uses detergents and mechanical force to separate seeded cells from their container. Eukaryotic cells are attached to the surface of a flask by cadherins. In the past, we’ve separated the cells from the flask by breaking these bonds with a protease, but in order to keep the proteins intact, a different method needs to be used to extract the proteins. In protein extraction for a western blot, we use detergents to lyse the membrane and free the proteins. The benefit of using a detergent is that it will dissolve the membranes and loosen proteins embedded in it. A cell scraper is also used to mechanically remove proteins and cells. Mechanical lysis would break open the membranes but wouldn’t free the membrane embedded proteins. One of the membranes broken open is the lysosomes. The lysosomes contain proteases that would destroy our target proteins. To compensate for this, we add a protease inhibitor. This will inhibit protein cutting and dephosphorylation of a few phosphorylated proteins, such as cyclin. To give the detergent time to fully dissolve the membrane, the solution was put on ice (figure 1) in order to slow down protein activity while the detergent breaks the membranes.
Standard Curve: To establish the concentration of the proteins isolated, it is compared against known concentrations in the form of a standard curve. The unknown concentration is made up of the extracted proteins and RIPA buffer, so known concentrations of BSA can be created in RIPA buffer. The known concentration’s absorbance readers can be read in a spectrophotometer, and graphed based concentration vs. absorbance. A standard line can be calculated and the line’s equation be interpreted (y=mx+b). Duplicate wells were used to reduce error in the standard line. The line’s equation can be used to estimate the concentration of the unknown. Substitute the absorbance of the unknown into the “y” of the equation and calculated
Protein Isolation: Protein isolation for a western blot uses detergents and mechanical force to separate seeded cells from their container. Eukaryotic cells are attached to the surface of a flask by cadherins. In the past, we’ve separated the cells from the flask by breaking these bonds with a protease, but in order to keep the proteins intact, a different method needs to be used to extract the proteins. In protein extraction for a western blot, we use detergents to lyse the membrane and free the proteins. The benefit of using a detergent is that it will dissolve the membranes and loosen proteins embedded in it. A cell scraper is also used to mechanically remove proteins and cells. Mechanical lysis would break open the membranes but wouldn’t free the membrane embedded proteins. One of the membranes broken open is the lysosomes. The lysosomes contain proteases that would destroy our target proteins. To compensate for this, we add a protease inhibitor. This will inhibit protein cutting and dephosphorylation of a few phosphorylated proteins, such as cyclin. To give the detergent time to fully dissolve the membrane, the solution was put on ice (figure 1) in order to slow down protein activity while the detergent breaks the membranes.
Standard Curve: To establish the concentration of the proteins isolated, it is compared against known concentrations in the form of a standard curve. The unknown concentration is made up of the extracted proteins and RIPA buffer, so known concentrations of BSA can be created in RIPA buffer. The known concentration’s absorbance readers can be read in a spectrophotometer, and graphed based concentration vs. absorbance. A standard line can be calculated and the line’s equation be interpreted (y=mx+b). Duplicate wells were used to reduce error in the standard line. The line’s equation can be used to estimate the concentration of the unknown. Substitute the absorbance of the unknown into the “y” of the equation and calculated