What Buffer Should Be Used to Dilute Boiled Protein Lysate for Western Blotting?

For Western blotting, boiled protein lysate can be diluted using one of the following buffers:

 

Protein Extraction Buffer

The extraction buffer used for initial protein isolation (e.g., RIPA buffer, PBS, or Tris buffer) can also be used for dilution, helping to maintain protein stability.

 

1X Loading Buffer

A 1X SDS-PAGE loading buffer, typically composed of SDS, β-mercaptoethanol, glycerol, bromophenol blue, and Tris-HCl, ensures proper protein migration during electrophoresis.

 

Dilution Procedure

1. Select an Appropriate Buffer

Choose either the protein extraction buffer or 1X loading buffer.

 

2. Adjust the Protein Concentration

The protein lysate is typically diluted to a final concentration of 1–2 µg/µL.

 

3. Mix Gently

To ensure homogeneity, mix the diluted protein solution carefully while avoiding excessive agitation, which may lead to protein aggregation.

 

MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.

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