Bio-Rad PROTEAN II XL Cell - Manual (Page 45)

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1. To prepare the monomer solutions, combine all reagents, except the APS and TEMED, and deaerate
under vacuum for ≥15 minutes. To initiate polymerization, add the APS and TEMED, and swirl gently to
mix.
2. Follow the instructions in Sections 3 and 4 for set-up and casting of the gels.
14.4 Running Conditions*
We recommend that gels be run under constant current conditions with an appropriate power supply, such as
the PowerPac 3000 or Model 1000/500 power supply (see Section 13.6 for ordering information).
Current Conditions mA per gel
Gel Thickness Stacking Gel Separating Gel
0.5 8 12
0.75 13 18
1.0 16 24
1.5 25 35
3.0 50 70
Run time is between 4 and 5 hours, depending on length of gel. Under constant current conditions, voltage
will gradually increase during the run. As a safety precaution, always set voltage, current, and power limits when
possible.
*Recommended power conditions for optimal resolution with minimal distortion.
14.5 Comparison of Coomassie Blue and Silver Staining
Coomassie Blue Silver Stain
Procedure: Sensitivity: Stain 1/2 hour with 0.1%
Coomassie Blue R-250 in
fixative (40% MeOH,
10% HOAc).
Destain with 40%
MeOH/10% HOAc to
remove background (usually
1 to 3 hrs).
14.6 2-D Stock Solutions
First Dimension IEF Tube Gels
Detergent Solution
0.3 g CHAPS
100 µl Nonidet P-40
900 µl ddH20
Dissolve CHAPS in water, then add Nonidet P-40 (NP-40).