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Measurement of acetyl-CoA
Last updated date: Mar 17, 2020 Views: 1416 Forks: 0
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-‐Grow 9 x 105 cells overnight in a 10 cm dish.
-‐Treat as desired.
-‐Rinse 1x with cold PBS
-‐Scrape into 1 ml cold PBS and transfer to 1.7 ml microfuge tube.
-‐Pellet cells in a microfuge at 4°C.
-‐Resuspend cells in 250 µl cold acetyl CoA assay buffer (from kit).
(Samples can be frozen at -‐80°C prior to further processing. Thaw on ice before use.)
-‐Add 24 µl cold 1M perchloric acid (PCA) and vortex.
-‐Let sit on ice 5 min.
-‐Spin 3 min at 16000 x g in a microfuge at 4°C.
-‐Transfer supernatant to a new tube.
(Save pellet for protein assay. Solubilize in 100-‐200 µl 0.2 N NaOH overnight at 37°C. Store at -‐20°C until assayed.)
-‐Precipitate PCA out of supernatant with 145 µl cold 2M KOH.
-‐Spin 15 min at 16000 x g in a microfuge at 4°C.
-‐Transfer supernatant to a new tube and check pH with pH paper. If pH is not between 6.5 and 8, then adjust using 1M PCA or 2M KOH as needed.
-‐Use 50 µl/well for acetyl CoA assay according to manufacturer's instructions (Sigma MAK039). You will need 200 µl total for each sample.
-‐Normalize acetyl CoA levels to protein levels determined using BCA assay on PCA pellet.
- Maher, P(2020). Measurement of acetyl-CoA. Bio-protocol Preprint. bio-protocol.org/prep252.
- Currais, A., Huang, L., Goldberg, J., Petrascheck, M., Ates, G., Pinto-Duarte, A., Shokhirev, M. N., Schubert, D. and Maher, P.(2019). Elevating acetyl-CoA levels reduces aspects of brain aging. eLife. DOI: 10.7554/eLife.47866
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