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1. Freeze flies to be assayed in a 1.5 ml microcentrifuge tube at -80°C for 15 min.
2. Add 100 μl of 250 mM Tris Buffer and homogenize or sonicate in sonicating water bath for 1 min.
3. Freeze the homogenate (-80°C) then thaw at room temperature.
4. Incubate at 65°C for 5 min.
6. Microcentrifuge at maximum speed for 10 min (see Hint #2).
7. Transfer the supernatant to a new microcentrifuge tube.
8. For quantitative assays, determine protein concentration (see Protocol on Protein Quantification) and add an equal amount of protein to each of the reaction tubes.
9. Prepare the CAT assay reaction as follows:
X μl of Drosophila extract (up to 90 μl)
1 μl of CAM
20 μl of 4 mM Acetyl CoA
Y μl of 250 mM Tris Buffer
Final reaction volume is 140 μl.
The amount of Drosophila extract added depends on the protein concentration. The remaining volume to 140 μl should be 250 mM Tris buffer.
10. Incubate for 37°C from 15 min to several hours (the incubation time should be determined empirically by doing a time course).
11. Stop the reaction and extract using 500 μl Ethyl Acetate.
12. Vortex well, microcentrifuge at maximum speed for 1 min and save the organic (top) phase.
13. Dry the organic extraction overnight in a fume hood (or concentrate using a speedvac).
14. Resuspend in approximately 15 μl Ethyl Acetate.
15. Spot Ethyl Acetate onto TLC plate (Baker-Flex TLC Silica Gel 1B plates, 20x20 cm).
16. Run in TLC buffer for 1.5 to 2.5 hr in a tightly sealed chromatography tank.
17. Dry the plates and expose at room temperature (follow the manufactures recommendations for exposure of plate).
18. Four spots should be observed. The lowest spot is non-acetylated CAM, the next two spots are two different monoacetylated CAM forms and the top spot (which may not be visible depending on the reaction duration) is diacetylated CAM.
19. For quantitative analysis, the spots can be cut out and counted on a scintillation counter.
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| TLC Buffer |
| 95 parts Chloroform
5 parts Methanol
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| 4 mM Acetyl CoA |
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| CAM |
| (CAUTION, see Hint #1)
0.2 μCi/μl 14C-Chloramphenicol
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| Tris Buffer |
| 250 mM Tris-Cl, pH 7.8
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Chloroform
Methanol
Ethyl Acetate
Acetyl CoA
Tris
14C-Chloramphenicol
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. Heat inactivates and precipitates most other proteins including some that inhibit CAT activity.
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