Hoefer SE900 - Manual (Page 33)

• p27
9. 1% Agarose in 1X Electrophoresis Buffer
(100 ml)
In a 500 ml flask add:
Agarose
1 g
10X Electrophoresis Buffer (Solution #8) 10 ml
Deionized H
2
O 90 ml
Bromophenol Blue 3 mg
Gently swirl to suspend agarose.
Heat at low power in a microwave oven until agarose is fully dissolved.
Divide into ~1.5 ml plastic screw top tubes.
Store in aliquots at 4 °C.
10. Gradient Gel Casting Displacement Solution
(50% Glycerol in 0.375 mM TrisCl 8.8, 0.1% SDS, 200 ml)
Glycerol
100 ml
1.5M TrisCl ph 8.8 (Solution #2) 50 ml
Deionized H
2
O 50 ml
Bromophenol Blue 3 mg
Mix well.
11. SDS Equilibration Buffer Solution
This solution is used after IEF, and before second dimension PAGE. The IPG
strips are immersed in excess solution to raise the pH of the strip buffer so
that it is suitable for PAGE, and to coat the proteins in SDS uniformly so
that they migrate properly in the second dimension gel.
Prepares 200 ml
(6 M urea, 75 mM Tris-HCl pH 8.8, 29.3% glycerol, 2% SDS, 0.002%
bromophenol blue)
Final Concentration Amount
Urea (FW 60.06) 6 M 72.1 g
1.5M Tris-HCl, pH 8.8 stock solution 75 mM 10.0 ml
Glycerol (87% w/w) 29.3% (v/v) 69 ml
SDS (FW 288.38) 2% (w/v) 4.0 g
Bromophenol blue 0.002% (w/v) 4 mg
Deionized H
2
O to 200 ml
Aliquot into 30 ml aliquots and store frozen at -20 °C or below.
24 cm IPG’s require 5 –10 ml per strip per equilibration step. Shorter strips
can use proportionately less volume per equilibrations step.
Caution! SDS may cause the solution to boil
over so exercise caution when heating and
prevent boiling over.
Note: IPG strips should be equilibrated
just prior to second dimensin PAGE.
Do not equilibrate the IPG strips before
storing at -20 °C.