mohammad tabish ahmed - eTheses Repository - University of ...

Chapter 2
2.6 Protein analysis
2.6.1- Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).
Reagents required:
- 1X Loading buffer (SDS-LB): 500µl of 0.05 M Tris-Cl (pH 6.8) was mixed with 2 ml
of 2% SDS, 1 ml of 10% Glycerol and 0.05g of bromophenol blue. The solution was
then made up to 10ml in MilliQ water and 500µl of β-Mercaptoethanol was added.
- Electrophoresis buffer: A stock solution of 5X electrophoresis buffer (1L) was made
with 15.1g of TRIS, 94g of glycine, and 5g of SDS made up to 1L in MilliQ water.
When running the gels a solution of 1X electrophoresis buffer was used. This
comprised 200µl 5x buffer added to 800µl MilliQ water.
- Staining solution: A 1L stock of staining solution was made with 2.5g of coomassie
brilliant blue R250, 450ml of ethanol, 450ml MilliQ, and 100ml of glacial acetic acid
mixed thoroughly to dissolve the coomassie powder.
- Destaining solutions: This comprised 300ml Methanol and 100ml glacial acetic acid
made up to 1L in MilliQ water.
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