Difference between revisions of "SDS sample buffer"
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Revision as of 16:42, 20 March 2013
For preparation and loading of protein samples onto a gel for SDS-PAGE analysis (Western blot/protein blot).
- Preparation: SDS contained in the sample buffer makes proteins negatively charged proportionally to their length. 2-mercapto-ethanol/DTT breaks disulphide bonds.
- Loading: glycerol makes the sample buffer more dense than the surrounding running buffer of the protein gel, enabling easy loading into the gel pockets.
To make 10 mL of 4x stock
- 2.0 ml 1M Tris-HCl pH 6.8
- 0.8 g SDS
- 4.0 ml 100% glycerol
- 0.4 ml 14.7 M β-mercaptoethanol
- 1.0 ml 0.5 M EDTA
- 8 mg bromophenol Blue
Final concentrations (1x)
- 50 mM Tris-HCl pH 6.8
- 2% SDS
- 10% glycerol
- 1% β-mercaptoethanol
- 12.5 mM EDTA
- 0.02 % bromophenol blue
Use of loading buffer
Dilute protein sample 1:3 into 4X sample loading buffer. Load on acrylimide gel in SDS-PAGE buffer.
β-mercaptoethanol is a severe irritant and is readily absorbed through the skin. SDS is a respiratory irritant in solid form and a mask should be worn while weighing it.