Lidstrom:Enzyme Assay Basics

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(Controls)
(Controls)
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** - enzymes + substrate  (strain = empty vector control or equivalent)
** - enzymes + substrate  (strain = empty vector control or equivalent)
** - enzymes - substrate  (strain = empty vector control or equivalent)
** - enzymes - substrate  (strain = empty vector control or equivalent)
-
* Mary likes to see a plot like [[image:sample_assay_data_format.jpg|thumb|center|upright=3.5|cartoon of a good way to depict enzyme assay data]]
+
* Mary likes to see a plot like [[image:sample_assay_data_format.jpg|thumb|center|upright=1.5|cartoon of a good way to depict enzyme assay data]]

Revision as of 17:58, 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, 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.

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
    cartoon of a good way to depict enzyme assay data
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