Western Blot Protocols-Sample & Gel Preparation

Western Blot Protocols-Sample & Gel Preparation Sample preparation is the first step and one of the most important steps of western blot. A good sample preparation makes your western blot half success. By understanding the nature of your starting sample and having a clear picture of the information you wish to derive from your Western blotting experiments, the chances of a successful analysis could be insured.

In principle, all sources of protein, from single cells to whole tissues as well as extracellular matrices, biological fluids and proteins secreted in vitro, are open to analysis by Western blotting. Whereas sources such as mammalian cells in suspension are easily disrupted under mild conditions and readily release their proteins, it is more difficult to extract proteins from intact tissues or within solid tumors. Extraction of the proteins from plants, bacteria and fungi are further complicated. Regardless of the source and protein of interest, however, the aim must be to harvest the proteins of interest, while at the same time, obtaining a sufficient yield of proteins at an acceptable level of purity.

General tips of sample preparation: Be gentle! Stay cool! • Use extraction procedures that are as mild as possible. Protect the proteins to keep them from denaturation. • Extract proteins quickly, on ice if possible and use buffer to maintain a suitable pH in order to prevent the protein degradation.