Lidstrom:Enzyme Assay Basics: Difference between revisions
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== Resources: == | |||
* [TONS of info from NIH & NCBI: http://www.ncbi.nlm.nih.gov/books/NBK53196/]. Some that may be of interest: | |||
** [http://www.ncbi.nlm.nih.gov/books/NBK92007/ Basics of Enzymatic Assays for HTS]. Though written for high-throughput screening, the principles apply to any enzyme assay. | |||
** [http://www.ncbi.nlm.nih.gov/books/NBK92001/ Mechanism of Action assays for Enzymes] | |||
== Cell Pellet Prep == | == Cell Pellet Prep == |
Revision as of 12:32, 1 November 2013
Back to Protocols
Resources:
- [TONS of info from NIH & NCBI: http://www.ncbi.nlm.nih.gov/books/NBK53196/]. Some that may be of interest:
- Basics of Enzymatic Assays for HTS. Though written for high-throughput screening, the principles apply to any enzyme assay.
- Mechanism of Action assays for Enzymes
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.
- If using NADH, the extinction coefficient at 340nm is 6220 M-1cm-1
- 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)
- Normalize for protein concentration.
- How to convert units from plate reader to mM/min or uM/min:
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
- 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.