Can the IP sample, which was left at room temperature for nearly a day (already boiled), still be used after forgetting to take it out and the instrument was turned off at night?

Consider the following key points:

1. Protein integrity

1. Samples that have been boiled (usually containing SDS and reducing agents) have most of their native conformation and enzymatic activity destroyed, theoretically preventing further significant degradation.

2. However, prolonged exposure at room temperature may lead to aggregation of components that are prone to precipitation or insolubility, especially when containing high concentrations of SDS or urea, which may precipitate upon cooling.

 

2. Microbial contamination and buffer systems

1. If the buffer does not contain antimicrobial agents (other than high concentrations of SDS), prolonged room temperature storage may still allow bacterial growth, leading to sample turbidity or degradation.

2. Buffer systems such as Tris may experience slight pH changes at room temperature, but these usually have minimal impact.

 

3. Subsequent use determines whether it can still be used

1. If only used for SDS-PAGE/Western blot: It can generally be used, but it is recommended to centrifuge to remove potential precipitates, and if necessary, re-boil.

2. If required for enzymatic experiments or mass spectrometry (especially quantitative mass spectrometry): Direct use is not recommended due to higher risks of contamination and modification (oxidation, deamidation), which may affect data reliability.

 

Suggested handling:

• Centrifuge first to check for visible precipitates or turbidity;

• If only performing WB, it can be used directly or re-boiled before use;

• If performing mass spectrometry or functional experiments, it is recommended to prepare anew, or at least make a control sample to assess the impact.

 

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