Does the Protein Degrade if Mixed with Loading Buffer for WB and Frozen at -80°C Without Prior Boiling for Denaturation?
In Western blot (WB) experiments, when protein is mixed with loading buffer but not boiled for denaturation, and is subsequently frozen at -80°C, protein degradation generally does not occur.
The loading buffer typically contains reducing agents (e.g., β-mercaptoethanol) and SDS (sodium dodecyl sulfate), which facilitate protein denaturation and dissociation. During WB experiments, protein samples are typically mixed with loading buffer and then boiled to denature the proteins and dissociate them into individual subunits.
Boiling denaturation ensures complete denaturation and dissociation, allowing the protein to migrate uniformly during gel electrophoresis. Without boiling, proteins may retain their native structure, resulting in uneven migration and affecting the accuracy of the WB results.
However, if the protein is mixed with loading buffer but not boiled, and then frozen directly at -80°C, degradation typically does not occur. Freezing at low temperatures slows protein degradation, and components in the loading buffer, such as SDS, may provide some protective effect.
It is important to note that prolonged freezing may cause structural changes or degradation of the protein. To ensure stability, it is recommended to perform boiling denaturation before freezing and to minimize freeze-thaw cycles during storage.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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