反应详情

Leupeptin and aprothinin (from Boerhinger) stock solutions (0.1%) for each weigh out 0.0025 g and add both to 2.5 mL of distilled water. Mix, aliquot and store at -20° C. for up to 6 months or store 1 week at 4° C.; Phenylmethylsulfonylfluoride stock solution (PMSF) 10 mg/mL, prepared from 0.05 g of PMSF and 5 mL of isopropanol (Store in the dark at room temperature for up to 1 year); Complete homogenation buffer (from Tris HCl/EDTA buffer (100 mL), PMSF stock (350 μL, final concentration of 35 μg/mL), and leupeptin/aprotinin stock (50 μL, final concentration of 0.5 μL/mL)); BSA buffer, prepared from complete homogenation buffer+2 mg/mL BSA, fraction V; n-butyryl co-enzyme A (Sigma B1508 lyophilized powder diluted to 5 mg/mL and stored at -20° C. in small aliquots to avoid excessive freeze-thawings); CAT enzyme (Sigma C8413 chloramphenicol acetyltransferase); 14C-chloramphenicol (NEN NEC-408A chloramphenicol, D-threo-[dichloroacetyl-1,2-14C]-CAT assay grade (TLC based), stored at 4° C. and diluted 1:10 in BSA buffer just prior to use); mixed xylenes (Aldrich); paraoxon (diethyl-n-nitrophenyl phosphate, from Sigma is a liquid with 90% purity) stock solution is prepared by diluting the original liquid 1:80 in isopropanol (Note: To achieve a final paraoxon concentration of 5 μM in a 130 μL of reaction mixture, a further 1:200 dilution in homogenization buffer is made and 3.3 μL of this 0.8 mM paraoxon solution is added to the reaction mixture); BCA protein reagent.

1. additionMix
2. waitaliquot and store at -20° C. for up to 6 months
3. waitstore 1 week at 4° C.
4. customBSA buffer, prepared from complete homogenation buffer+2 mg/mL BSA, fraction V
5. customstored at 4° C.
6. additiondiluted 1:10 in BSA buffer just
7. customis prepared
8. customin a 130 μL of reaction mixture