Measurement of acetyl-CoA

-­‐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.

Category