The Blotting Sandwich: A Crucial Step in Western Blotting Procedures

When it comes to western blotting, the preparation of a sample lysate is a crucial step that requires careful attention to detail. In this article, we will delve into the world of blotting sandwiches and explore how they play a vital role in separating proteins by their molecular weight rather than their native conformational shape or charge.

The term 'blotting sandwich' might seem unusual, but it's a fitting description for the combination of ingredients that come together to create a perfect blend of denaturation, separation, and visualization. The key players in this sandwich are bromophenol blue, glycerol, SDS, beta-mercaptoethanol or DTT, and a loading buffer such as Laemmli sample buffer.

To create the blotting sandwich, each sample is first vortexed and then incubated at 95 degrees Celsius for five minutes to completely denature the proteins. This step is essential in preparing the proteins for separation by their molecular weight. The denaturation process allows the proteins to unwind and lose their native conformational shape or charge, making it possible to separate them based on their size.

The blotting sandwich is then complete with the addition of bromophenol blue to visualize the lysate, an ionic buffer to maintain a stable pH, glycerol to allow the samples to sink into each well, SDS to assist in denaturing and provide a net negative charge to the protein, and beta-mercaptoethanol or DTT to reduce disulfide bridges between cysteines.

Reduced and denatured conditions are typically used for Western blots, allowing proteins to be separated by their molecular weight rather than their native conformational shape or charge. This is crucial in identifying specific proteins of interest and understanding their role in various biological processes.

In addition to the blotting sandwich, other essential steps in western blotting procedures include tissue preparation, cell lysis, and centrifugation. Cell lysis is a critical step that requires the use of a suitable lysis buffer containing protease inhibitors to prevent protein degradation. Freshly prepared protease inhibitors should always be used, and samples should be kept on ice and worked quickly to minimize contamination.

When it comes to choosing a lysis buffer, the localization of the protein of interest is a key consideration. Proteins localized in the cytoplasm can be solubilized using relatively mild extraction detergents, whereas proteins bound to membranes require stronger extraction detergents.

In conclusion, the blotting sandwich is an essential component of Western blotting procedures that allows for the separation of proteins by their molecular weight rather than their native conformational shape or charge. By carefully combining denaturation agents, buffers, and protease inhibitors, scientists can unlock valuable information about specific proteins and their role in various biological processes. Whether you're a seasoned researcher or just starting out in the world of Western blotting, understanding the importance of the blotting sandwich is crucial for successful protein analysis.

Blotting sandwich