SDS-PAGE sample buffer (Morris formulation)
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 -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?
