ACAT in vitro assay

From LipidomicsWiki

In vitro ACAT assay from crude cell lysate

Solutions needed:
1xPBS
Buffer A (20mM Tris-HCl, 1mM EDTA; pH7.7)
Buffer A + 10mg/ml Fatty acid free BSA + 50µg/ml cholesterol (10µl/sample)
Buffer A + 2mg/ml Fatty acid free BSA + 1µCi/ml [14C]oleoyl CoA (50µl/sample)

Sonicate the solutions with Fatty acid free BSA on water bath for 30 seconds and let stand at +37oC for 30 minutes

PREPARATION OF CELL LYSATES:
The following steps are done at +4oC
1. Wash cells with 1xPBS and scrape into eppendorf tubes
2. Spin down cells, 2000 x g 5 min, +4oC
3. Resuspend pellet in Buffer A (150µl), let stand for 5 minutes on ice
4. Homogenize with a 25gauge needle, 10 passes
5. Take 20µl aside for protein determination and proceed immediately with assay (protein concentration should be ~2-5µg/µl)

ANALYSIS:
1. Mix 40µl lysate with 10µl Buffer A containing 10mg/ml FAF-BSA and 50µg/ml cholesterol
2. Incubate at +37oC for 2 minutes
3. Start reaction by adding 50µl Buffer A containing 2mg/ml FAF-BSA and [14C]Oleoyl CoA
4. Vortex briefly and incubate at +37oC for 10 min
5. Stop reaction by adding 1ml CHCl3 and 1ml MetOH
6. Add ca 10000 dpm of [3H]cholesteryl oleate as an internal standard
7. Take 1/10 of mix to determine initial radioactivity in the samples
8. Add 1 ml H2O and 1 ml CHCl3 vortex, let stand, mix again
9. Centrifuge for 10 minutes at 2500 rpm
10. Take the lower phase into a KK-tube
11. Evaporate the solvent with N2
12. Dissolve the lipid films into ~30 μl of CHCl3/MeOH 9:1 mix
13. Pipet to TLC along with unlabeled cholesterol ester standard and run with hexane/diethyl ether/acetic acid 80:20:1 solvent
14. Stain the standard with iodine vapour and scrape the cholesteryl ester bands into scintillation tubes. Count with dual label program, correct for procedural losses according to 14C-standard and plot against the amount of protein in the sample.