DFR Assay (for DHK, DHQ & DHM)
In 1 mL:
0.1M Tris (pH 7.4)
1µmol substrate (= 0.304mg DHQ, 0.288mg DHK, 0.32mg DHM, 0.286mg eriodyctiol: dissolve
in >95% MeOH. Our Sigma naringenin is racemic, so use 2µmol, 0.54mg)
1µmol NADPH (= 1 vial ~ 1mg; I generally dissolve this in the Tris)
6µmol glucose-6-P (= 1.7mg) (benchtop)
1 U glucose-6-P dehydrogenase (= 0.25 mg) (-20C in dessicator; this is an enzyme)
200-400 µg enzyme (I use 250uL of bacterial extract)
Mix and incubate 3 hr at 30ºC
(adjust volumes accordingly if doing ½ reactions for timepoints):
Add 15uL 6N HCl to kill the reaction
Immediately extract 3X in 1mL ethyl acetate (I shake these vigorously and then spin down ca
30s in the centrifuge. EtAc is the top layer and contains the leucoanthocyanidins, which you
want).
Wash twice with 200µL H2O (H2O forms a small bubble at the bottom- try to get as much as
possible out)
Evaporate EtAc under vacuum at 35-45ºC into ~ 100µL aqueous phase
For leucoanthocyanidins, add 1mL Butanol-HCl reagent (95:5 v/v) and heat at 95ºC for 30 min
(historically, we have used n-butanol but t-butanol is preferable to reduce ether formation at
the 3-carbon)
For flavan-4-ols, add 1mL 2N HCl and heat at 95ºC for 30 min, then extract into isoamyl to
clean, if desired