Tips for Detecting Phosphoproteins by Western Blot
Protein phosphorylation is very important for protein post-translational modification that controls many cellular processes including metabolism, transcriptional and translation regulation, degradation of proteins, cellular signaling and communication, proliferation, differentiation, and cell survival . Approximately 35% of human proteins are phosphorylated. Phosphoproteins are low in abundance, and, therefore, are, sometimes, challenging to detect and characterize by Western blot and other methods. Western blot is the most common method for detecting and quantifying phosphorylated proteins. Western for phosphoprotein is not really different from a regular Western blot but following factors need pay attention to in order to obtain consistently results. Although all factors are important, protein sample preparation is possibly the most critical and deserve in-depth exploring.
Keep phosphoproteins intact. Once cells are lysed, protease and phosphatase are immediately released into the cell lysate. The turnover rate of phosphatases is very rapid. A phosphorylated protein can be dephosphorylated in a matter of milliseconds . To prevent proteins from being degraded and dephosphorylated, the cell lysate must be kept on ice. Protease and phosphatase inhibitor cocktails must also be added to cell lysis buffer prior to use. Soon after the protein concentration of cell lysate is determined, the cell lysate should be mixed with SDS-PAGE loading buffer as soon as possible because the loading buffer can also inhibit phosphatase activity.
Avoid Using Milk for Blocking. Milk contains casein, an abundant phosphoprotein that can cause high background when milk is used to block the membrane. When doing your Western blot, you should use an alternative blocking solution such as bovine serum albumin, or other non-milk blocking agent such as WB Blocking Solution (Cat# WA-008, Invent Biotechnologies).
Optimize Experimental Conditions. Phosphorylation is a key response to cellular signaling and the phosphorylation status of a protein changes dynamically. Some proteins are phosphorylated only under certain conditions such as chemical or biological stimulation. You need to optimize your conditions to find out when your protein is phosphorylated. For example, you may need to stimulate the cells prior to harvesting. Try different stimulation conditions and do time courses to find when the highest level of phosphorylation is achieved .
Multiplex the Phosphorylated Proteins and Have Proper Controls. A simple way to look at total protein versus phosphorylated protein is to look at both proteins on the same blot using fluorescent antibodies, this is also known as multiplexing. Using the right combination of secondary antibodies, you can easily examine and determine the phosphorylation state of a protein. Always include a control in which you probe for the