preparation of gel
buffer
Laemmli buffer
colloidal coomassie staining
aquaeous coomassie staining



separating gel (5 ml, one minigel, 0,75 mm spacer)


 6%
 8%
 10%
 12%
water
 3
 2,75
 2,5
 2,25
1,5M Tris pH 8,8, 0,4% SDS
 1,25
 1,25
 1,25
 1,25
PAA (40%)
 0,75
 1
 1,25
 1,5
TEMED
 3
 3
 3
 3
APS (10%)
 50
 50
 50
 50

overlay the gel with 2-Propanol during polymerisation

stacking gel: (1,5 ml, one mini gel)

921 µl water
375 µl 0,5 M Tris pH 6,8, 0,4% SDS
187 µl 40% PAA
1,5 µl TEMED
15 µl  APS


large gels (16x16cm, 1mm spacer)
set up the glass plates and spacers according to Dobberstein's procedure. Prepare about 50 ml for the separating gel and 10 ml for the stacking gel.


Buffer for stacking gel
  0,5M Tris HCl pH 6,8, 0,4% SDS

Buffer for separating gel
1,5 M Tris HCl pH 8,8, 0,4% SDS

Acrylamid stock
40% with 29,1 : 0,9 Bis, Merck

APS
make 10% stocks in water, freeze at -20°C

Laufpuffer (10x, 1l)

150 g Glycin
10 g SDS
32,8 g Tris base
ad 1l water

Staining solution (500 ml)

200 ml Methanol (tech.)
200 ml water
80 ml acetic acid (tech.)
0,5 g Coomassie R-250 (0,1% w/v)
staining solution may be reused, pour back after staining

Destaining solution (2000 ml)

600 ml methanol (tech.)
200 ml acetic acid (tech.)
ad 2000 ml water

barrels of technical grade solvents (methanol, ethanol, acetic acid)  are stored in the basement (Lösungsmittelkeller).

Laemmli buffer (SDS-PAGE sample buffer)

  • 2x    0,09 M Tris-HCl pH 6,8, 6% SDS, 0,6% bromophenol blue, 20% Glycerol, 6% ß-mercaptoethanol
  • 5x     0,225 M Tris/HCl pH6,8, 15% SDS, 1,5% bromphenolblue, 50% glycerol, 15% (v/v) ß-mercaptoethanol
  • may be complemented with 8 M urea

Electrophoresis:

mini gel:  20 mA, about 1h

large gels:  60 mA, with ventrilation, ca. 2-4h


Colloidial coomassie staining

necessary for MS analysis of bands, less sensitive than silver stain

  • incubate gel in 3% (v/v) acidic acid
  • equilibrate gel in solution B, discard solution
  • add 200 ml of solution B, 2 ml of solution C, incubate for 24h, RT
  • wash in solution B
  • fix staining in solution A, 2 x 30 min
  • very briefly rinse the gel with 100% ethanol (techn), removes crystals from the surface of the gel, will also remove staining of bands
  • wash 2x with tap water
  • store gel in solution A
solution A:  200g/l ammonium sulfate in 1% phosphorous acid
solution B:  60 g/l ammonium sulfate in 1% phosphorous acid
solution C:  2% (w/v) coomassie G450 in 50% ethanol

Aquaeous coomassie staining

necessary for gel purification of antigens for immunisation
  • run a large SDS-PA gel
  • incubate in 0,5% Coomassie blue R-250 in water overnight
  • save staining solution
  • wash several times with water, destaining takes some time