Yu:SDS PAGE: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Michaelcyu (talk | contribs) (sds-page casting) |
(No difference)
|
Revision as of 15:13, 13 June 2009
SDS-PAGE
Casting Gel
1. Assemble the gel-casting apparatus | Assemble casting apparatus | ||||||||||||||||||||||||||||||
2. In a 15mL tube, prepare resolving gel for the desired percentage: | Mix Resolving Gel | ||||||||||||||||||||||||||||||
| |||||||||||||||||||||||||||||||
3. Add 50μL 10% APS and pour resolving gels immediately. APS catalyzes 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 | ||||||||||||||||||||||||||||||
| |||||||||||||||||||||||||||||||
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 to the stacking gel in the tube. Pour stacking gels immediately, as APS catalyzes 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 |