ProtoGel Resolving Buffer (4X)

Measure and Mix Solutions:
The volume of ProtoGel required for gel casting solutions of any volume and acrylamide concentration may be calculated from the following formula:

V_p =(X) (V_t)/40

where:

V_p = Volume of 40% ProtoGel
X = % Monomer Desired in Gel
V_t = Total Volume of Gel Casting Solution

EXAMPLE: To make 100 ml of a 10% monomer gel, calculate the volume of ProtoGel to add as follows:
V_p = (10) (100)/40 = 25.0 ml

Initiate and Cast Gel:
For optimal results degas gel solution for 10 minutes under vacuum aspiration prior to innitiation with APS and TEMED. Add 1.0ml of 10% (w/v) ammonium persulfate for every 100ml of gel casting solution. Swirl gently to mix. Add 0.1 ml of TEMED for every 100ml of gel casting solution. Swirl gently to mix. Pour the solution into the gel casting cassette. The gel should begin to set in 10-20 minutes. To provide a sharp interface, overlay the gel with water saturated  n-butanol during polymerization. Flush butanol away with water just before casting the stacking gel (below).

Cast Stacking Gel:
Use ProtoGel Stacking Buffer to make 10ml of a 4% stacking gel:

• ProtoGel: 1.00ml
• ProtoGel Stacking Buffer: 2.50ml
• Deionized water: 6.44ml

Add 0.05ml 10% ammonium persulfate and 0.01ml of TEMED. Gel will begin to set in 20 minutes.