Lidstrom:Enzyme Assay Basics

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(Example of Preparing for an assay that monitors NADH consumption)
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* Determine how fast the reaction proceeds in the absence of your enzymes at various substrate concentrations.
* Determine how fast the reaction proceeds in the absence of your enzymes at various substrate concentrations.
** Example: look at Vmax as you increase [formate] for an assay that has formate as a substrate and NADH as the cofactor and substance you are monitoring.  You should have an increase in Vmax as you increase [formate] because there are formate dehydrogenases present.  As you increase [formate] you will saturate these enzymes (curve 1 in the picture below).  You may see that increases in [formate] lead to decreases in Vmax (curve 2 in the cartoon below); this is caused by inhibition.  You want to chose a value of formate that is high enough to saturate the background FDHs if you want to observe the rate caused by enzymes you are adding.
** Example: look at Vmax as you increase [formate] for an assay that has formate as a substrate and NADH as the cofactor and substance you are monitoring.  You should have an increase in Vmax as you increase [formate] because there are formate dehydrogenases present.  As you increase [formate] you will saturate these enzymes (curve 1 in the picture below).  You may see that increases in [formate] lead to decreases in Vmax (curve 2 in the cartoon below); this is caused by inhibition.  You want to chose a value of formate that is high enough to saturate the background FDHs if you want to observe the rate caused by enzymes you are adding.
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[[image:131031_add_more_formate.jpg|thumb|center|upright=1.5|cartoon of adding increasing [formate] to a strain not expressing special enzymes.]]
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[[image:131031_add_more_formate.jpg|thumb|center|upright=1.5|cartoon of adding increasing [formate] to a strain not expressing the enzymes you are testing for.]]

Revision as of 18:26, 31 October 2013

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Contents

Cell Pellet Prep

General guidelines:

  • 50 mL of E. coli in LB/TB is usually plenty. (stationary phase)
  • If using a methylotroph, you may need more culture volume. Some strains are "sick" and don't grow very turbid. Use:
    • 100 mL of at OD 0.6-0.8 ~or~
    • 200 mL at OD = 0.4 ~or~
    • 300 mL at OD = 0.2

Of course the amount of biomass depends on how well your enzyme is expressed and what its specific activity is.

Lysis

  • Use a similar mass of cells for different strains you are breaking.
    • This allows for more accurate BCA results. JM (10/2013) finds strong dependency on protein concentration calculations depending on the dilution used when dilutions span an order of magnitude.
  • Resuspend in 2 mL of an appropriate lysis buffer
  • French press 2-3 times.
  • Centrifuge out debris. Perhaps ultracentrifuge.

Analyze protein concentration

  • 1000 ug/mL is good, says Ceci

Assay cells

  • Ceci/Amanda always do 200 uL in assays. There is no reason not to do 100 or 150 uL though. JM (10/2013)
  • Ceci adds 50 uL per rxn. She said you don't want to dilute the enzymes more than you need to, as they are most happy when concentrated.
    • This may require that you use more substrate. Hopefully your substrate is cheap.
    • If you are doing an NADH-linked assay, there is a limit to the amount of NADH you can add, as you will saturate the spectrophotometer.

Calculating Enzyme Rates

  • The slope provided by the plate reader needs to be converted to moles/time/mg protein or similar units.
  • Convert absorbance to M
    • If using NADH, the extinction coefficient at 340nm is 6220 M-1cm-1
      • If there is less volume in the well, the conversion factor is greater because the path length is larger.
  • Factor in the path length.
    • If using the crystal plate in the plater reader, 200 uL is 0.51 cm. (Elizabeth "Betsy" Skovran figured this out)

Controls

  • The basic set of tests you should do:
    • + enzymes + substrate
    • + enzymes - substrate
    • - enzymes + substrate (strain = empty vector control or equivalent)
    • - enzymes - substrate (strain = empty vector control or equivalent)
  • Mary likes to see a plot like
    cartoon of a good way to depict enzyme assay data.  Each bar is Vmax for reaction with substrate - Vmax for rxn without substrate.
    cartoon of a good way to depict enzyme assay data. Each bar is Vmax for reaction with substrate - Vmax for rxn without substrate.
  • You could also subtract the empty vector height from the control height, but Mary said she prefers to see them separately. JM 10/31/2013

Example of Preparing for an assay that monitors NADH consumption

  • First, determine how fast NADH oxidizes in your assay environment. It is pH and buffer dependent. It may not be zero.
  • Determine how fast the reaction proceeds in the absence of your enzymes at various substrate concentrations.
    • Example: look at Vmax as you increase [formate] for an assay that has formate as a substrate and NADH as the cofactor and substance you are monitoring. You should have an increase in Vmax as you increase [formate] because there are formate dehydrogenases present. As you increase [formate] you will saturate these enzymes (curve 1 in the picture below). You may see that increases in [formate] lead to decreases in Vmax (curve 2 in the cartoon below); this is caused by inhibition. You want to chose a value of formate that is high enough to saturate the background FDHs if you want to observe the rate caused by enzymes you are adding.
cartoon of adding increasing [formate] to a strain not expressing the enzymes you are testing for.
cartoon of adding increasing [formate] to a strain not expressing the enzymes you are testing for.
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