Burkart:HPLC CoAAssay

Overview
Uses the HPLC to analyze the activity of your CoA biosynthetic enzymes (PanK, PPAT, DPCK), based on the characteristic HPLC shift to lower retention time on addition of negative charge to the CoA intermediate.

Materials
Stock Solutions
 * 250 mM ATP (151 mg/mL in 1M Tris-HCl pH 8)
 * 0.5 M KPO4 (pH 7.4)
 * 1M KCl
 * 2M MgCl2
 * CoA Biosynthetic Enzymes (His or MBP)
 * 200 mM KC12 (coumarin-pantetheine)

Master Mix (1 mL - enough for 33x30 uL rxns)
 * 420 uL H20
 * 400 uL 0.5M KPO4 (final conc 200 mM)
 * 80 uL 1M KCl (final conc 80 mM)
 * 80 uL 0.25M ATP (final conc 20 mM)
 * 20 uL 2M MgCl2 (centrifuge after adding - final conc 40 mM)
 * 5 uL 0.2M KC12 (final conc. 1 mM)

Procedure
For a 30 uL reaction:


 * 1) add 30 uL Master mix see recipe above) to 1.5 mL epi tubes (x4)
 * 2) to each epi add:
 * 3) *2.5 uL H2O
 * 4) *0.5 uL PanK + 1.5 uL H2O
 * 5) *0.5 uL PanK + 1 uL PPAT + 1 uL H2O
 * 6) *0.5 uL PanK + 1 uL PPAT + 1 uL DPCK
 * 7) incubate RT 30 min.
 * 8) *during this time turn on HPLC - equilibrate column (Burdick and Jackson OD5 w/ guard column front loaded - 25 cmx4.6 mm) with buffer
 * 9) *use method JLM_BUFA.M - uses buffer A = 25 mM KPO4 pH 6.55, buffer B = acetonitrile (NO TFA)
 * 10) *0-5 min isocratic A, 5-20 min gradient to 45% B, 20-28 min gradient to 100% B, 28-29 min gradient to 100%A, 29-35 min 100%A1
 * 11) transfer rxns to HPLC tubes
 * 12) *10 uL reactions - monitor @ 220 nm, 254 nm (adenine), 360 nm (coumarin)
 * 13) *coumarin pantetheine: 20.88 min
 * 14) *coumarin phosphopantetheine: 17.15 min
 * 15) *coumarin dephospho-CoA: 16.02 min
 * 16) *coumarin CoA: 14.68 min

Contact

 * jmeier@ucsd.edu

or instead, discuss this protocol.