Bio-Rad PROTEAN II XL Cell - Manual (Page 46)

40
First Dimension Capillary Tube Gel Monomer Solution
11 g urea (9.2 M final concentration)
3 ml acrylamide/bis stock (4.5% total monomer)
Stir to dissolve the urea. This step is done slowly without heating or with very gentle heating only.
0.2 ml Bio-Lyte
® 5/7 ampholyte
0.8 ml Bio-Lyte 3/10 ampholyte
1 ml detergent solution (CHAPS/NP-40) from above
Add deionized water to 20 ml.
Mix and degas.
Add 20 µl TEMED after degassing to avoid evaporation.
Add 40 µl 10% (w/v) APS, swirl 8-10 times and cast the gels.
This makes 20 ml total volume, enough to cast one set of gels using the casting tube.
Sample Solution A
1.0 g SDS
0.232 g DTT or DTE
Dissolve in ddH2O to a final volume of 10 ml. Store in aliquots at -70 °C. Iso-Urea Solution E
0.1 g DTT
0.4 g CHAPS
5.4 g urea
500 µl Bio-Lyte 3/10 ampholyte
6 ml ddH2O
Electrolytes
First Dimension Upper Running Electrolyte-Cathode (20 mM NaOH)
Dissolve 0.4 g NaOH in 500 ml deionized water and degas thoroughly for 30 minutes.
First Dimension Lower Running Electrolyte-Anode (10 mM H3PO4)
Dilute 1.8 ml concentrated H
3
PO
4
in 2.6 liters deionized water and degas thoroughly for 30 minutes.
Transfer Solution
40 ml 0.5 M Tris-HCl, pH 8.8
80 ml 10% SDS
8 ml 0.05% bromophenol blue
150 ml ddH
2
O
14.7 Running Conditions
IEF is carried out at 200 volts constant voltage for 2 hours, followed by 500 volts constant voltage for 2 hours,
and finally 800 volts constant voltage for 16 hours (overnight).