Burkart:HPLC CoAAssay: Difference between revisions

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==Overview==
==Overview==
IN PROGRESS!!


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.
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.
Line 17: Line 15:
Master Mix (1 mL - enough for 33x30 uL rxns)
Master Mix (1 mL - enough for 33x30 uL rxns)
*420 uL H20
*420 uL H20
*400 uL KPO4 (final conc 200 mM)
*400 uL 0.5M KPO4 (final conc 200 mM)
*80 uL KCl (final conc 80 mM)
*80 uL 1M KCl (final conc 80 mM)
*80 uL ATP (final conc 20 mM)
*80 uL 0.25M ATP (final conc 20 mM)
*20 uL MgCl2 (centrifuge after adding - final conc 40 mM)
*20 uL 2M MgCl2 (centrifuge after adding - final conc 40 mM)
*5 uL KC12 (final conc. 1 mM)
*5 uL 0.2M KC12 (final conc. 1 mM)


==Procedure==
==Procedure==
#Step 1
For a 30 uL reaction:
#Step 2
 
#*Step 2 has some additional information that goes with it. i.e. Keep at 4°C.
#add 30 uL Master mix see recipe above) to 1.5 mL epi tubes (x4)
#Step 3
#to each epi add:
##Step 3 has multiple sub-steps within it.
#*2.5 uL H2O
##Enumerate each of those.
#*0.5 uL PanK + 1.5 uL H2O
#*0.5 uL PanK + 1 uL PPAT + 1 uL H2O
#*0.5 uL PanK + 1 uL PPAT + 1 uL DPCK
#incubate RT 30 min.
#*during this time turn on HPLC - equilibrate column with buffer
#*use method JLM_BUFA.M - uses buffer A = 25 mM KPO4 pH 6.55, buffer B = acetonitrile (NO TFA)
#*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
#transfer rxns to HPLC tubes
#*10 uL reactions - monitor @ 220 nm, 254 nm (adenine), 360 nm (coumarin)
#*coumarin pantetheine: 20.88 min
#*coumarin phosphopantetheine: 17.15 min
#*coumarin dephospho-CoA: 16.02 min
#*coumarin CoA: 14.68 min
 


==Notes==
==Notes==
Line 37: Line 48:
#*omit KCl in KPO4 buffer (Worthington special)
#*omit KCl in KPO4 buffer (Worthington special)
#*works fine in Tris-Cl - must include KCl
#*works fine in Tris-Cl - must include KCl
#*pH 7.5 seems optimal - CoaE won't turnover positively charged CoA analogues (i.e. amino pantetheine) unless at pH 9 or higher
#*reaction tolerates pH 6.5-8.0 - 7.4 seems optimal - CoaE won't turnover positively charged CoA analogues (i.e. amino pantetheine) unless at pH 9 or higher
#*for prep scale (> 1 mL) chemoenzymatic CoA synthesis I have used the following conditions in the past: 400 mM Tris 7.4, 50 mM ATP, 4 mM pantetheine analogue, 20 mM KCl, 15 mM MgCl2, 60 uL/mL each CoA biosynthetic enzyme (PPAT, DPCK, PanK)
#always add MgCl2 last to master mix or will precipitate in neutral/basic buffered sol'n  
#always add MgCl2 last to master mix or will precipitate in neutral/basic buffered sol'n  
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.

Revision as of 18:13, 22 April 2009

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:
    • 2.5 uL H2O
    • 0.5 uL PanK + 1.5 uL H2O
    • 0.5 uL PanK + 1 uL PPAT + 1 uL H2O
    • 0.5 uL PanK + 1 uL PPAT + 1 uL DPCK
  3. incubate RT 30 min.
    • during this time turn on HPLC - equilibrate column with buffer
    • use method JLM_BUFA.M - uses buffer A = 25 mM KPO4 pH 6.55, buffer B = acetonitrile (NO TFA)
    • 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
  4. transfer rxns to HPLC tubes
    • 10 uL reactions - monitor @ 220 nm, 254 nm (adenine), 360 nm (coumarin)
    • coumarin pantetheine: 20.88 min
    • coumarin phosphopantetheine: 17.15 min
    • coumarin dephospho-CoA: 16.02 min
    • coumarin CoA: 14.68 min


Notes

  1. this is Jordan's mix - I've seen a lot off diff. reagent/enzyme conc work in this assay - key is to find one that looks for you
  2. other conditions you may have luck with:
    • omit KCl in KPO4 buffer (Worthington special)
    • works fine in Tris-Cl - must include KCl
    • reaction tolerates pH 6.5-8.0 - 7.4 seems optimal - CoaE won't turnover positively charged CoA analogues (i.e. amino pantetheine) unless at pH 9 or higher
    • for prep scale (> 1 mL) chemoenzymatic CoA synthesis I have used the following conditions in the past: 400 mM Tris 7.4, 50 mM ATP, 4 mM pantetheine analogue, 20 mM KCl, 15 mM MgCl2, 60 uL/mL each CoA biosynthetic enzyme (PPAT, DPCK, PanK)
  3. always add MgCl2 last to master mix or will precipitate in neutral/basic buffered sol'n
  4. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  5. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers and books

  1. Worthington AS and Burkart MD. One-pot chemo-enzymatic synthesis of reporter-modified proteins. Org Biomol Chem. 2006 Jan 7;4(1):44-6. DOI:10.1039/b512735a | PubMed ID:16357994 | HubMed [Worthington-OBC]
  2. Meier JL and Burkart MD. Chapter 9. Synthetic probes for polyketide and nonribosomal peptide biosynthetic enzymes. Methods Enzymol. 2009;458:219-54. DOI:10.1016/S0076-6879(09)04809-5 | PubMed ID:19374985 | HubMed [Meier-Meth-Enz]

All Medline abstracts: PubMed | HubMed

Contact

  • jmeier@ucsd.edu

or instead, discuss this protocol.


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