I am trying to calculate the amount of peptide I would need to order to complete a bacterial protease activity assay using fluorescence. I hoping to look at E. coli and S. aureus strains with specific protease - peptide activity.
Would there be a suggested amount of peptide to order to be able to complete activity assays in triplicate?
Hello..
The amount of peptide you need to order for a bacterial protease activity assay using fluorescence will depend on several factors, including the specific assay conditions you plan to use, such as the concentration of bacterial cells, the concentration of peptide, and the duration of the assay.
As a starting point, a typical concentration of bacterial cells used in protease activity assays is around 10^8 cells/mL. The peptide concentration can vary depending on the specific activity of the protease and the specific activity of the assay, but a typical concentration range is between 0.01-10 μM.
To perform the assay in triplicate, you would need to order enough peptide to perform the assay three times. So, you would multiply the volume of the assay (let's say 1 mL) by the concentration of the peptide (let's say 1 μM) and multiply it by the number of assays. You will have the amount of peptide you need in micrograms to perform this.
It would be best to consult the protocol or paper you will use as reference, that should indicate the specific concentrations and conditions used in the study, so you will have a better idea of how much peptide you will need.
It is also important to consider an extra amount of peptide in case of any losses during handling and storage, also if your final goal is to test different strains and different proteases, you will probably want to order extra peptide to account for variability in the assay results, since you plan to run different samples.
I hope this information helps!
Regards.
The above answers are both correct. You need to ensure that the substrate concentration is well above the Km for the protease. You also need to fix the pH and temperature, which means your measurements won't be at Vmax for many of the proteases, but the answer will be consistent across samples. Finally, the growth conditions for the cells will affect the results. Protease activity increases as the cells exit exponential growth and move into the stationary phase. The type of nutrient exhaustion will affect the results with protease activity in Carbon > Nitrogen > Phosphate starvation. I would suggest trying a published colorimetric protease assay e.g., trypsin activity first to get an approximate range for the protease activity.
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