Western blotting – loading & running buffers
SDS-PAGE loading buffer (loading dye)
SDS-PAGE loading buffer / loading dye is used for loading protein samples on a protein gel and monitoring the gel run. To make 10mL of 5 X SDS-PAGE loading buffer mix the following:
50mM Tris pH 6.8 (1mL from a 0.5M stock)
2% SDS (2 mL from a 10% stock. When handling SDS powder wear a face mask and/or work in the fume hood).
10% glycerol (1mL from a 50% stock. A 50% stock in water is easier to pipette).
deionised water to a final volume of 9mL. (The final 1mL is reserved for DTT to be added immediately before use).
bromophenol blue 2 drops
Store at room temperature. Prior to use add fresh DTT to a final concentration of 100mM (1:10 dilution from a 1M frozen stock). 5 X SDS-PAGE loading buffer should be used at a ratio of 1:4 (buffer : protein sample). When running the gel, run bromophenol blue to the bottom of the gel.
SDS-PAGE running buffer
To make 1L of 10 X SDS-PAGE running buffer (for filling gel running tanks) mix the following:
14.4 g glycine
3.05 g Tris
0.1% SDS (When handling SDS powder wear a face mask and/or work in the fume hood).
deionised water to a final volume of 1L
Mixing with a magnetic stirrer is ideal. Store at room temperature. For use at 1 X concentration make a 1L bottle by mixing 100mL of 10 X buffer with 900mL of deionised water.