SDS PAGE electrophoresis
Including:
Sample preparation
Electrophoresis assembly
SDS-PAGE electrophoresis
Materials:
SDS PAGE Loading Buffer
To make 10 mL of 4x stock:
2.5 ml 1M Tris-HCl pH 6.8
1g SDS
0.5 ml 0.3% Bromophenol Blue
4 ml of neat glycerol
MilliQ water up to 10 ml
Before use: add 50ul of neat β-mercaptoethanol into 1 ml of the stock solution
4X concentrations
250mM Tris-HCl pH 6.8
10% SDS
0.005% Bromophenol Blue
5% (approx. 0.7M) β-mercaptoethanol
40% Glycerol
Final concentrations (1x)
62.5mM mM Tris-HCl pH 6.8
2.5% SDS
0.00125 % Bromophenol Blue
1.25% (approx. 0.18M)1% β-mercaptoethanol
10% Glycerol
5x Running buffer 5L :
75g Tris
360g Glycine
25g SDS
MilliQ water up to 5L
Protein markers:
PageRuler Plus Pre-stained protein ladder x2 (Rainbow marker) or
Protein Marker, Broad Range (P7702S)
Protocols:
Sample preparation:
Mix your samples with loading buffer. Usually 5ug protein to up to 15ul MilliQ
water and 5ul loading buffer.
Boil them at 90 °C for 15 min or samples may also be treated at 37 °C for 2 h.
Electrophoresis assembly
Make sure a complete gelation of the stacking gel and take out the comb.
Take the glass plates out of the casting frame and onto the electrode
assembly and set them in the tank.
Pour 1x Running Buffer into the inner chamber and in the outer chamber and
remove the combs.
SDS-PAGE electrophoresis
Load prepared samples into wells and make sure not to overflow. Don't forget
loading protein marker into the first lane.
Attach the power leads and apply 100 volts to run the electrophoresis until the
blue dye front reaches the bottom.
.
Note: Various factors affect the properties of the resulting gel.
 Higher concentration of ammonium persulfate and TEMED will lead to a faster gelation, on the
other hand, a lower stability and elasticity.

The optical temperature for gel gelation is 23°C-25°C. Low temperature will lead to turbid,
porous and inelastic gels.

The pH is better to be neutral and the gelation time should be limited in 20-30 min.