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
  • 1gSDS
  • 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:

PageRulerPlus 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 MilliQwater 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.

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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.