SDS-PAGE sample buffer (Morris formulation)

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Contents

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 is used to denature proteins and make them negatively charged. In this manner each protein will migrate in the electroporetic field in a measure proportional to its lenght.
  • 2-mercaptoethanol is used to break disulphide bonds
  • Glycerol increases the density of the sample relative to the surrounding runnig buffer making it easier to load in the well
  • Bromophenol blue is used to follow the run of protein sample 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 -20C.
  • Add the appropriate volume of a β-mercaptoethanol 100% stock to your samples just before denaturing them at 95C.

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 95C. 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 95C. 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 it is easily adsorbed through skin.
  • β-mercaptoethanol

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

  • The Morris SDS-PAGE protein sample loading buffer has been used by Caterina Strambio De Castillia in the Blobel and in 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.


Contact

  • Who has experience with this protocol?

caterina.strambio@umassmed.edu

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