Lidstrom:Enzyme Assay Basics
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Revision as of 17:51, 31 October 2013
Back to Protocols
Cell Pellet Prep
- 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.
- 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
- 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.
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)