How to Prepare the WB Sample Loading Buffer?

In Western Blot (WB) experiments, the sample loading buffer (Loading Buffer or Sample Buffer) typically contains the following key components:

 

1. Tris-HCl Buffer

0.5 M, pH ~6.8

 

2. SDS (Sodium Dodecyl Sulfate)

10%

 

3. Glycerol

20-30%

 

4. Bromophenol Blue

0.1-0.2% (tracking dye)

 

5. β-Mercaptoethanol or DTT (Dithiothreitol)

5-10%

 

Preparation and Usage

Mix the above components in the desired proportions, adjusting the final volume as needed. Typically, the loading buffer is mixed with the sample at a 1:1 or 1:3 ratio, followed by boiling for 5-10 minutes for denaturation. After cooling to room temperature, load the sample into the SDS-PAGE gel wells for electrophoresis.

 

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