When to boil protein in WB experiments?
"Boiled Protein" (also known as "boiled sample") refers to heating a protein sample at high temperatures for a period of time to achieve protein denaturation. This typically involves mixing the protein-containing sample with a loading buffer containing SDS and a reducing agent (such as β-mercaptoethanol or DTT) and heating it in a boiling water bath for 5-10 minutes.
Generally, before SDS-PAGE gel electrophoresis, once the sample extraction is completed and mixed uniformly with the loading buffer, it can undergo heat denaturation. After heat denaturation, the sample can be immediately used for SDS-PAGE, followed by Western Blot.
It should be noted that certain sensitive or easily degradable proteins may not require prolonged heating or may be heated at slightly lower temperatures. However, for most proteins, conventional boiling treatment is effective and necessary.
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