Why should proteases and inhibitors be added during protein extraction for WB experiments?
The addition of proteases and inhibitors during the protein extraction process in Western Blot (WB) experiments serves the following purposes:
1. Addition of Proteases:
1. The presence of proteases might degrade the target proteins, so adding proteases during protein extraction can prevent protein degradation.
2. Proteases can degrade intracellular proteins, including receptor proteins on the cell membrane, cytoskeletal proteins, etc. By adding proteases, these intracellular proteins can be degraded, reducing non-specific background signals in the WB experiment.
2. Addition of Inhibitors:
1. During protein extraction, cells may release some endogenous proteases, which could degrade the target proteins. Adding inhibitors can effectively inhibit these endogenous proteases, protecting the integrity of the target proteins.
2. Inhibitors can block intracellular signaling pathways, preventing abnormal signal transduction during the extraction process and maintaining the original state of the proteins.
The addition of proteases and inhibitors in WB experiments helps protect the integrity of target proteins and reduce non-specific background signals, thereby improving the accuracy and reliability of the experiments.
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