Protocol for Reverse Zymograms
Separating/Reverse Zymo Gel
3.75 ml 4X Tris-Cl, pH 8.8
3.25 ml distilled water
6.50 ml Protogel (30% mix 29:1 acrylamide:bisacrylamide (3.3% C) Biorad 161-0156
1.5 ml gelatin (1% solution)
20 ul gelatinase A (Chemicon CC071; 0.1 mg/ml)
100 ul 10% APS
6 ul TEMED
Use a pasteur pipette to slowly pour gel between glass plates up to mark on glass plates. Layer with ddH2O, and rock back and forth to ensure that interface is even. Leave 15-20 min to polymerize.
3.4 ml ddH2O
0.63 ml 0.5M Tris (pH6.8)
0.83 ml 40% acrylamide
50 ul 10% SDS
50 ul 10% APS
5 ul TEMED
Pour off water from top of gel. Use a pasteur pipette to load stacking gel on top of separating gel. Carefully insert combs between plates, making sure there are no bubbles on comb. Top of well heads on the comb should be submerged. Leave 30 min to polymerize.
After the gel has polymerized, snap gels into the gel apparatus, one on either side. It helps to prewet the gaskets with running buffer, this makes the chamber more leakproof.
4X Tris-Cl/SDS, pH 8.8
91 g Tris base
300 ml H2O
Adjust pH to 8.8
Add 2g SDS
Add water to 500 ml total volume.
Filter and store at 4C.
5X Running Buffer
Trizma base 9g
SDS 3g or 30 ml of 10% SDS
dH2O to 600 ml
Store at 4C.
Dilute the stock 5X running buffer to 1X running buffer: 100 ml 5X stock to 400 ml ddH2O. Fill the inner buffer reservoir to the top of the inner glass plate.
1. Load standards and samples. We use pre-stained SDS-PAGE Lo-Range MW standards from Biorad (19-101 kD). Load 7.5 ul of standard in the first well. Load 10- 20 ug of each sample (based on total protein content as determined by protein assay.
2. Place gels into the tank, fill outer buffer chamber with the remaining 1X running buffer. Use syringe with bent needle to flush bubbles away from bottom of gel.
3. Run gel on constant voltage 100V for 5-10 minutes, until the samples line up at the edge of the separating gel. Turn voltage up to 200V, and run samples until dye front is 4 -5 mm from bottom of gel.
4. Wash gel 2 times 30 min in 2.5% Triton X100 with shaking.
5. Place gels in rubbermaid dishes and cover with zymogram incubation buffer. Leave overnight at 37C. You may vary incubation times to determine optimum activities for each sample.
6. Wash in 40 ml 10% TCA for 10 min. at r.t. with shaking. To make 80 ml 10% TCA - add 16 ml 50% TCA to 64 ml ddH2O.
7. Mix 20 ml destain, 20 ml ddH2O and 2 ml coomasie blue rapid stain for each gel. Stain in this solution for 30 minutes with shaking.
8. Destain in full strength destain solution. Shake in destain a few minutes by hand, until you have clear bands on a dark blue background.
9. Photograph gels on a light box using an orange 15 filter, shutter speed 1/15 s, aperature F32, Polariod 667 Film.
10. Store the gel in destain diluted 1:1 with ddH2O.