Team:Caltech/Protocols/PABA HPLC assay

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=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. 
* Column size ? 

==Procedure==

===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.

[[Image:PABA Standard HPLC assay - 08-07-08.PNG|thumb|center|pABA eluting at 4.9-5.0 minutes for the standard curve.]]

===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).

[[Image:08-07-08_pABA_Standard_Curve_plot.jpg | thumb|center |pABA HPLC standard curve with 1:3 dilutions starting from 10ug/ml pABA. The integral of the peak is plotted against the known pABA amount.]]

==Sources==
Guo-Fang Zhang, Kjell A. Mortier, Sergei Storozhenko, Jet Van De Steene, Dominique Van Der Straeten, Willy E. Lambert. ''Free and total para-aminobenzoic acid analysis in plants with high-performance liquid chromatography/tandem mass spectrometry.'' '''Rapid Communications in Mass Spectrometry''': Volume 19, Issue 8 , Pages 963 - 969, 2005.