Acetic acid detection kit

Acetate standard curves were made by diluting the Ricca acetic acid standard with HDAC8 assay buffer (above). The acetic acid detection kit (R-biopharm) was used according to the instructions except that the reaction volume and coupled solution volume were decreased 10-fold and no additional water was added to dilute the reaction. Solutions 1, 2, 3 and 4 in the kit were preincubated at room temperature for 20 minutes before being mixed with acetate. The reaction was incubated at room temperature for 40 minutes and then NADH fluorescence (ex. = 340 nm, em. = 460 nm) was measured using a Polarstar Galaxy fluorometer in a 96 well plate.

ATP, Coenzyme A, NAD⁺, L-malic acid, citrate synthase (CS), and malate dehydrogenase (MDH) were purchased from Sigma. The acetic acid detection kit was purchased from R-biopharm. Fluor de Lys peptide and the developing reagent were purchased from Enzo Life Sciences. The unlabeled peptides (Ac-KGGAKac-COO⁻, Ac-KGGAKac-NH₂, and Ac-KGGAKacW-NH₂) were purchased from Peptide2.0 (>85 % purity). Cobalt and magnesium were purchased as ICP standards from GFS Chemicals and the acetic acid standard was purchased from the Ricca Chemical Company. Chelex 100 resin was purchased from Bio-Rad. HDAC3/NCOR1 was purchased from Enzo Life Sciences. Ethylenediaminetetraacetic acid (EDTA) was purchased from Sigma-Aldrich at >99 % purity. All other materials were purchased from Fisher and were of a purity >95 % unless otherwise noted.