Team:Caltech/Protocols/PABA HPLC assay

{{Caltech_iGEM_08| Content=

para-Aminobenzoic Acid (pABA) HPLC Assay Protocol

Purpose
To test for para-aminobenzoic acid levels in E. coli overexpressing the pABA synthesis genes pabA and pabB.

Materials

 * Buffer A: 0.1% formic acid in H2O
 * Buffer B: 0.1% formic acid in MeOH
 * A high performance liquid chromatography machine.
 * HPLC Column: Eclipse XDB-C18 column, 5um particles, 4.6x150mm column, with attached guard column (all from Agilent)

Sample Prep

 * 1) Centrifuge the cell culture max speed, 10 minutes.
 * 2) Separate pellet from supernatant.
 * 3) Resuspend the pellet in 1mL 0.1M Tris-HCl buffer
 * 4) Sonicate the resuspended pellet for 1 minute, alternating between 0 and 12Hz.
 * 5) Filter sterilize the cell lysate and the supernatant.
 * 6) Load 500uL of each into the HPLC.

HPLC Method

 * 1) Injection volume was 20uL, column temperature was kept at 40°C.
 * 2) Flush the lines individually with Buffer A and B for two minutes each, flush the column with 92% A, 8% B for 10 minutes.
 * 3) From the source: "The starting eluent was 92% A mixed with 8% B; the proportion of B was increased linearly to 50% in 7 min, then to 100% in 3 min. The mobile phase was then immediately adjusted to its initial composition and held for 4 min in order to re-equilibrate the column."
 * 4) We found the retention time of pABA to be 4.9-5.0 minutes, however the literature states the retention time as 6.0 minutes.



Standard Curve
1. Run 1:3 dilutions starting from 10ug/ml pABA in wild type bacterial lysate using the same protocol to generate a linear standard (see below).



Contacts
Victoria Hsiao }}