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Casting Gel

Casting 2 SDS-PAGE Gels with ProtoGel
1. Assemble the gel-casting apparatus Assemble casting apparatus
2. In a 15mL tube, prepare resolving gel for the desired percentage: Mix resolving gel
Resolving Gel
12% 10% 8% 7%
ProtoGel 4mL 3.3mL 2.7mL 2.3mL
Millipore H2O 2.7mL 3.3mL 4mL 4.4mL
1.5M Tris pH 8.8 2.4mL
10% SDS 100μL
3. Add 50μL 10% APS and 10μL TEMED and pour resolving gels immediately. APS and TEMED catalyze the polymerization of the gel Add 50μL 10% APS and pour gel
4. Pour resolving gels to approximately 2-3 cm below the edge of the gel cassette. The space above is required for stacking gel
5. Add a thin, .5 cm layer of isopropanol to the gel cassettes immediately after pouring the resolving gel. This ensures that the top edge of the resolving gel is level, free of bubbles, and polymerizes evenly Add isopropanol
6. While the resolving gel is polymerizing, prepare stacking gel in a 15mL tube: Mix stacking gel
Stacking Gel (5%)
ProtoGel 650μL
Millipore H2O 3.2mL
1.5M Tris pH 6.8 475μL
10% SDS 37.5μL
7. Wait for the resolving gel to fully polymerize. When the leftover stacking gel in the tube has solidified and tilting the casting apparatus from left to right does not cause the gel to shift, polymerization is complete
8. Gently wash isopropanol out of the gel cassette with millipore H2O and dry with a kimwipe Remove isopropanol after polymerization
9. Add 25μL 10% APS and 5μL TEMED to the stacking gel in the tube. Pour stacking gels immediately, as APS and TEMED catalyze polymerization Add 25μL 10% APS and pour
10. Add a comb containing the desired number of wells immedietely. Ensure that no bubbles or air pockets are trapped under the comb Add gel comb
11. Allow stacking gel to fully polymerize before using. Gels can be stored wrapped in damp paper towels in a 4°C refrigerator for a week or more

Loading Gel

Loading SDS-PAGE gels with samples
1. Assemble SDS-PAGE running apparatus, with SDS-PAGE Buffer Assemble gel apparatus
2. Prepare samples for loading. Add loading buffer if necessary, and boil if necessary Prepare samples for loading
3. Using gel loading pipette tips, load 12-15μL of sample into each lane. Include any markers or benchmarks. Load any empty lanes with 1x gel loading buffer to ensure lanes run straight Load samples

Running Gel

Running SDS-PAGE gels
1. Attach electrodes to the appropriate terminals and run at 100V for approximately 20 minutes to stack samples Run at 100V for 20 minutes to stack
2. Transfer the apparatus to a cold room or add an icepack to prevent overheating and run at 170V until the bottom of the lane is about to run off the gel Run at 170V in cold room to resolve