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detecting phosphorylated proteins on a Western blot is very similar to running a regular Western blot the basic technique is the same sds-page transfer and detection however there are a few tips and tricks to consider when detecting phosphorylated proteins here are six tips that can help you obtain the best results as much as possible make sure you keep your samples and buffers on ice endogenous proteases and phosphatases in your life States will digest and de phosphorylate the proteins in your samples at warmer temperatures to reduce the chances of dephosphorylation use optimize lysis buffers and add appropriate phosphatase inhibitors block the membrane in BSA when running a Western blot of phosphorylated proteins a milk based blocking buffer is not suited for the Western blot of a phosphorylated protein as it contains casein a phosphorylated protein as well as a high level of phosphatases that can cleave the phosphate off of the target protein make sure you select a primary a
