Team:Caltech/Protocols/PABA HPLC assay

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=para-Aminobenzoic Acid (pABA) HPLC Assay Protocol=
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<font face="verdana" style="color:#CC3300">para-Aminobenzoic Acid (pABA) HPLC Assay Protocol</font></div>
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__TOC__
==Purpose==
==Purpose==
To test for para-aminobenzoic acid levels in ''E. coli'' overexpressing the pABA synthesis genes ''pabA'' and ''pabB''.
To test for para-aminobenzoic acid levels in ''E. coli'' overexpressing the pABA synthesis genes ''pabA'' and ''pabB''.
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* Buffer B: 0.1% formic acid in MeOH
* Buffer B: 0.1% formic acid in MeOH
* A high performance liquid chromatography machine.  
* A high performance liquid chromatography machine.  
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* Column size ?
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* HPLC Column: Eclipse XDB-C18 column, 5um particles, 4.6x150mm column, with attached guard column (all from Agilent)
==Procedure==
==Procedure==
===Sample Prep===
===Sample Prep===
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1. Centrifuge the cell culture max speed, 10 minutes.  
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# Centrifuge the cell culture max speed, 10 minutes.  
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# Separate pellet from supernatant.  
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2. Separate pellet from supernatant.  
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# Resuspend the pellet in 1mL 0.1M Tris-HCl buffer
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# Sonicate the resuspended pellet for 1 minute, alternating between 0 and 12Hz.  
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3. Resuspend the pellet in 1mL 0.1M Tris-HCl buffer
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# Filter sterilize the cell lysate and the supernatant.  
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# Load 500uL of each into the HPLC.
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4. Sonicate the resuspended pellet for 1 minute, alternating between 0 and 12Hz.  
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5. Filter sterilize the cell lysate and the supernatant.  
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6. Load 500uL of each into the HPLC.
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===HPLC Method===
===HPLC Method===
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1. Injection volume was 20uL, column temperature was kept at 40°C.  
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# Injection volume was 20uL, column temperature was kept at 40°C.  
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# Flush the lines individually with Buffer A and B for two minutes each, flush the column with 92% A, 8% B for 10 minutes.  
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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.  
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# 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."  
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# 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.
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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."  
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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.
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[[Image:PABA Standard HPLC assay - 08-07-08.PNG|thumb|center|pABA eluting at 4.9-5.0 minutes for the standard curve.]]
[[Image:PABA Standard HPLC assay - 08-07-08.PNG|thumb|center|pABA eluting at 4.9-5.0 minutes for the standard curve.]]
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[[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.]]
[[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.]]
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==Sources==
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===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.
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.
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===Contacts===
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[[User:vhsiao|Victoria Hsiao]]
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Latest revision as of 20:55, 15 October 2008


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para-Aminobenzoic Acid (pABA) HPLC Assay Protocol


Contents

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)

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.
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).

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.

Contacts

Victoria Hsiao

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