When to Boil the Protein in WB Experiments
"Protein denaturation by heat, often referred to as 'boiling the sample,' involves heating a protein sample at high temperatures to induce denaturation. Typically, this process includes mixing the protein sample with a loading buffer containing SDS and a reducing agent like β-mercaptoethanol or DTT, followed by heating in a boiling water bath for 5-10 minutes.
After completing the sample extraction and thorough mixing with the loading buffer, heat-induced denaturation is generally carried out prior to SDS-PAGE gel electrophoresis. Once denatured by heat, the sample can promptly be utilized for SDS-PAGE and subsequently for Western Blot analysis.
It is important to acknowledge that certain sensitive or easily degradable proteins may require shorter heating times or slightly reduced temperatures. Nonetheless, for the majority of proteins, this conventional boiling method is both effective and essential.
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