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Detecting phosphorylated proteins on a Western blot is similar to a regular Western blot with the same basic technique of sds-page transfer and detection. However, there are tips to consider for best results. Keep samples and buffers on ice to prevent protein dephosphorylation. Use optimized lysis buffers with phosphatase inhibitors. Block the membrane in BSA instead of milk-based buffer to avoid phosphatase interference. Select a primary antibody specific to phosphorylated proteins.
