SDS-PAGE sample buffer (Morris formulation)-PDF

Overview

This buffer is used for the preparation and loading of protein samples onto a gel for SDS-PAGE analysis.

• SDS contained in the sample buffer denature proteins and make them negatively charged. In this manner, each protein will migrate in the electrophoretic field proportionately to its length.
• β-mercaptoethanol is used to break disulfide bonds
• Glycerol increases the density of the sample relative to the surrounding running buffer making it easier to load in the well
• Bromophenol blue is used to follow the run of protein samples on the gel

Preparation

To make 10 ml of 4x stock

Mix the following:

• 2.5 ml 1 M Tris-HCl pH 6.8
• 0.5 ml of ddH20
• 1.0 g SDS
• 0.8 ml 0.1% Bromophenol Blue
• 4 ml 100% glycerol
• 2 ml 14.3 M β-mercaptoethanol (100% stock)

Adjust the final volume to 10 ml with ddH20

Final concentration (1x)

• 62.5 mM Tris-HCl pH 6.8
• 2.5 % SDS
• 0.002 % Bromophenol Blue
• 0.7135 M (5%) β-mercaptoethanol
• 10 % glycerol

Variant

To make 10 ml of 10x stock

• In 70 % glycerol / 30 % water, dissolve the following:

• • 0.606 g Tris-base
  • 2.5 g SDS

• Adjust the pH using pH indicator strips to 6.8
• Add 2 mg of Bromophenol Blue and make sure the powder is completely dissolved
• Adjust the final volume to 10 ml with 70 % glycerol / 30 % water before storing at -20°C.
• Add the appropriate volume of a β-mercaptoethanol 100% stock to your samples just before denaturing them at 95°C.

Use of the loading buffer

4x variant

Dilute the 4x loading buffer 1:3 in your sample. Denature proteins by heating samples for 10 minutes at 95°C. Load on SDS-PAGE and run.

10x variant

Dilute the 10x loading buffer 1:9 in your sample. Dilute β-mercaptoethanol 1:19 in your sample (i.e. 5% final concentration). Heath samples for 10 minutes at 95°C. Load on SDS-PAGE and run.

Safety

• Use a mask when you weigh out SDS powder.
• Use gloves when you handle β-mercaptoethanol as it is a serious irritant and is easily adsorbed through the skin.
• β-mercaptoethanol

References

• The Morris SDS-PAGE protein sample loading buffer has been used by Caterina Strambio De Castillia in the Blobel and the Rout laboratories in the period between 1992 and 2005.
• The original reference describing this formulation is at the moment not available. It will be posted as soon as it is.