How long is the protein re-boiling temperature and time? Can it be boiled after dilution?

Re-boiling protein samples (i.e., heat denaturation) is usually used before SDS-PAGE loading to disrupt the tertiary structure of proteins, ensuring that electrophoretic migration primarily reflects molecular weight. Specific re-boiling temperature and time are as follows:

 

I. Standard Re-boiling Conditions

• Temperature: 95°C or 100°C (boiling water)

• Time: 5 minutes

If the sample is viscous or highly concentrated, containing membrane proteins or other complex components, the time can be extended to 10 minutes, but avoid excessive boiling that may lead to protein degradation.

 

II. Can be boiled after dilution

Yes, but pay attention to the following points:

1. Ensure the Buffer system is intact

The diluted sample must still contain reducing agents (such as DTT or β-mercaptoethanol) and SDS to ensure full denaturation and reduction. Otherwise, boiling has little significance.

 

2. The dilution factor should not be too large

Excessive dilution may result in a protein concentration that is too low, affecting band clarity; it is generally recommended that post-dilution concentration remains >0.5 μg/μL.

 

3. It is not recommended to store for a long time after boiling

Samples that have been re-boiled are recommended to be loaded as soon as possible; especially systems containing SDS and reducing agents are prone to degradation or precipitation.

 

Biotech Pack -- Leading Service Provider for Biological Product Characterization and Multi-group Biological Mass Spectrometry Testing

 

Related Services:

Protein Immunoblotting and Electrotransfer Service