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sotellme
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should we add a reducing agent (DTT or 2-Mercaptoethanol) or not in the SDS_PAGE's sample buffer? The plan is using this gel for Western blot.
sotellme said:should we add a reducing agent (DTT or 2-Mercaptoethanol) or not in the SDS_PAGE's sample buffer? The plan is using this gel for Western blot.
The reducing agent helps to break down protein disulfide bonds, allowing for the separation and detection of individual protein subunits during SDS-PAGE and Western blotting.
Some commonly used reducing agents include dithiothreitol (DTT), β-mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine (TCEP).
The amount of reducing agent needed will vary depending on the specific experimental conditions and the protein being analyzed. Generally, a concentration of 5-10 mM is sufficient.
It is recommended to add the reducing agent to the SDS-PAGE sample buffer immediately before use, as it can lose its effectiveness over time when exposed to air.
In some cases, the reducing agent may cause protein degradation or interfere with downstream applications. It is important to optimize the concentration and duration of reducing agent treatment for each experiment to minimize these effects.