Separating Proteins in Cell Biology Lab Using Western Blotting

Western blotting is a powerful tool used in cell biology labs to separate and detect specific proteins within a sample. In this step, we will focus on preparing our sample lysate for loading onto an SDS page gel. To achieve this, we will use a positive electrode to attract negatively charged proteins.

The first step involves vortexing each sample and incubating it at 95 degrees Celsius for five minutes to completely denature the proteins. This process is crucial in ensuring that the proteins are unfolded and separated based on their molecular weight rather than their native conformational shape or charge.

To load our samples onto the gel, we will mix them with a loading buffer that contains several components. These include bromophenol blue, which helps to visualize the lysate; glycerol, which allows the samples to sink into each well; SDS, which assists in denaturing and provides a net negative charge to the protein; beta-mercaptoethanol or DTT, which reduces disulfide bridges between cysteines. By diluting each sample with a loading buffer such as Laemmli sample buffer, we can ensure that our proteins are separated based on their molecular weight.

Western blots are typically performed under reduced and denatured conditions to minimize the influence of native conformational shape or charge on protein separation. In this case, we will use the supernatant from our lysate, which contains the sample we want to analyze further.

Before preparing our samples for western blotting, we must first lyse our cells using a lysis buffer that contains protease inhibitors. This is essential in preventing the degradation of the protein of interest during the preparation process. The choice of lysis buffer depends on the localization of the protein of interest, with stronger extraction detergents required for solubilization of membrane-bound proteins compared to isolated cytoplasmic proteins.

To ensure optimal results, it's crucial to use freshly prepared protease inhibitors and work quickly when preparing our samples. We will start by adding ice-cold PBS and lysis buffer such as RIPA buffer, which is commonly used for maximum protein yield.

In summary, western blotting involves several key steps, including tissue preparation (preparation of sample lysate), loading the samples onto an SDS page gel, and analyzing the resulting blot. By following these procedures carefully, we can successfully separate and detect specific proteins in our cell biology lab using western blotting.

Cell biology lab western blot results