Team:TU Darmstadt/Protocols/SDS-Page

Contents 1 SDS-Page 1.1 About 1.2 SDS-PAGE (Laemmli) 1.2.1 Procedure 1.2.2 Stacking Gel 1.2.3 Separating Gel 1.2.4 Buffer 1.2.4.1 Running buffer 1.2.4.2 Staining buffer 1.2.4.3 Sample buffer 1.3 SDS-PAGE (Schägger) 1.3.1 Procedure 1.3.2 Stacking Gel 1.3.3 Separating Gel 1.3.4 Buffer 1.3.4.1 Cathode buffer 1.3.4.2 Anode buffer 1.3.4.3 Staining buffer 1.3.4.4 Sample buffer 1.4 Application 1.5 References

SDS-Page

About

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis is a technique to separate proteins according to their electrophoretic mobility (a function of the length of a polypeptide chain and its charge).^[1]

SDS-PAGE (Laemmli)

Procedure

• prepare the separating gel and fill it into the chamber
• pour 1 mL isopropyle alcohol on the top of the gel to destroy air bubbles and prevent dehydration
• discard the isoprpyl alcohol and pour the prepared stacking gel
• stick in the comb
• store the gel in wet cloth (to prevent dehydration) at 4°C

Stacking Gel

For two gels

• 0.5 mL glycine
• 1.25 mL 0.5 M Tris buffer pH 6.8
• 0.025 mL 20% SDS
• 0.67 mL acrylamide
• 0.005 mL TEMED
• 0.025 mL APS
• 2.575 mL dist. water

Separating Gel

For two gels

• 3.0 mL glycine
• 7.5 mL 1.5 M Tris buffer pH 8.8
• 0.15 mL 20% SDS
• 12.0 mL acrylamide
• 0.02 mL TEMED
• 0.15 mL APS
• 7.2 mL dist. water

Buffer

Running buffer

• 3,03 g TRIS-HCl
• 15 g glycine
• 0,2 g SDS
• ad. 1 L dist. water

Staining buffer

• 7 mg coomassie brillant blue
• 200 mL acetic acid
• 1 L ethanol
• ad. 2 L dist. water

Sample buffer

• 4.8 g urea
• 0.242 g TRIS-HCl
• 0.2 g SDS
• 1 mg sodium bromphenolate
• ad. 10 mL dist. water

SDS-PAGE (Schägger)

Procedure

• same as above

Stacking Gel

For five gels

• 1.6 mL acrylamide
• 12.4 mL Tris-HCl/0,3% SDS pH 8.45
  • 182 g TRIS-HCl
  • 1,5 g SDS
  • ad. 500 mL
  • adjust pH 8.45
• 7.6 mL dist. water
• 100 µL 10% ammonium persulfate (APS)
• 10 µL TEMED

Separating Gel

For five gels

• 20.00 mL acrylamide
• 20.00 3 M Tris-HCl/0.3%SDS pH 8.45
  • 182 g TRIS-HCl
  • 1,5 g SDS
  • ad. 500 mL dist. water
  • adjust pH 8.45
• 100 µl 10% ammonium persulfate (APS)
• 10 µL TEMED

Buffer

Cathode buffer

• 12.11 g TRIS-HCl
• 17.92 g tricine
• 1 g SDS (0.1% SDS)
• ad. 1 L dist. water

Anode buffer

• 24.22 g TRIS-HCl
• ad. 1 L dist. water
• adjust to pH 8.9

Staining buffer

• 7 mg coomassie brillant blue
• 200 mL acetic acid
• 1 L ethanol
• ad. 2 L dist. water

Sample buffer

• 4.8 g urea
• 0.242 g TRIS-HCl
• 0.2 g SDS
• 1 mg sodium bromphenolate
• ad. 10 mL dist. water

Application

• SDS-Page

References

[1] http://en.wikipedia.org/wiki/SDS-page