SDS-PAGE sample buffer (Morris formulation)

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Variant)
Current revision (15:16, 26 March 2013) (view source)
(Overview)
 
Line 4: Line 4:
* 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.
* 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
+
* β-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
* 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
* Bromophenol blue is used to follow the run of protein sample on the gel

Current revision

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.
  • β-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 -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 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-De-Castillia

Personal tools