Mary Mullins

CAT ASSAY: DROSOPHILA TRANSFORMANTS

CAT assay;
1. Freeze flies to be assayed in an Eppendorf tube (1.5 ml) at -80°C for 15 min.
2. Add l00 µl of 0.25M Tris (pH 7.8) and homogenize.
3. Freeze the homogenate, thaw, and sonicate (optional) for 1 minute.
4. Heat at 65°C for 5 minutes. This precipitates most other proteins including some inhibitor(s) of CAT activity.
5. Spin in cold (rm. temp O.K.) microfuge for 10 minutes.
6. Transfer supernatant to new tube. For quantitative assays, do Bradford assays on all extracts so equal amounts of protein can be used in each assay.
7. Set up reactions:
Extract: 1-90 µl
0.25M Tris-7.8 + 14C-chloramphenicol(CAM): 30 µl=29 µl Tris + 1 µl
CAM (0.2µCi)
4mM Acetyl CoA; (Lithium;) : 20µl
0.25M Tris: adjust final volume to 140µl
8. Incubate at 37°C from l5 minutes to several hours. For good quantitation, a time course can be done to determine the linear range of the assay.
9. Stop the reaction and extract the chloramphenicol by adding 0.5ml ofethyl acetate. Vortex well and spin 1 minute in microfuge. Remove the top organic phase and place in new tube.
10. Dry in Speedvac (about 15 minutes) or overnight in the hood.
11. Resuspend in about l5 µl ethyl acetate.
12. Spot onto TLC plates (American Scientific Products, Baker-Flex TLC Silica Gel 1B Plates, 20x20cm, Cat. # 4462).
13. Run in 95:5 chloroform:methanol for l.5-2.5 hours. in a tightly sealed chromatography tank.
14. Dry plate and expose at room temp.
15. Four spots may be seen. The lowest is the non-acetylated CAM;, the next two spots are two different monoacetylated CAM forms, the top spot ( which may not be seen depending on the duration of the reaction) is diacetylated CAM. For quantitative assays, the spots can be cut out and counted in a scintillation counter.
Materials:
€14C-Chloramphenicol(CAM): Amersham, CFA515, 54mCi/mmole