Hi,
I am trying to develop a method for CYP2C9 inhibition by some compounds. I wanted to use flourescent based assays for this. So, I decided to use Dibenzylfluorescein (DBF) for this assay. I have referred to some literature for this (https://www.sigmaaldrich.com/US/en/product/sigma/d7191). However, when I tried to develop the method, I was having difficulties. The enzyme was metabolizing the substrate very quickly.
I am attaching some of my graphs for this with experimental conditions. I would be grateful if someone can suggest me how to improve my assay conditions.
Step 1: Enzyme 1-8 mg/ml (https://www.sigmaaldrich.com/US/en/product/sigma/mtoxce2c9?context=product)
Master mix :NADP - 2.6 mM, NADPH 2.6 mM, Glucose-6-phosphate dehydrogenase 0.8 u/ml, Glucose-6-phosphate 6.6 mM, MgCl2 6.6 mM)
Step 2: Mix enzyme at concentration to master mix - add 30 ul of each solution to well of 384 well plate
Step 3: prepare DBF solution (DBF stock 2 or 4 mM in acetonitrile) - final DBF concentration between 1-4 uM - add 30 ul in each well with the enzyme
Step 4: shake the plate in orbital mode in plate reader
Step 5 read at 425 eX and 438 Em
I am getting the graph which looks as attached. Please let me know how can I improve.
I must mention that since I am doing kinetic study, I do not add NaOH at the end.
Regards,
Pratik
Here are some comments.
1. I don't understand why you have NADP in your assay mix. You shouldn't need that.
2.The emission wavelength should be 538 not 438 nm.
3. Dissolve the DBF in a more enzyme-compatible and less volatile solvent than acetonitrile, preferably DMSO. Keep the DMSO concentration no higher than 2% by volume in the final assay mixture.
4. Initiate the reaction by adding the 2 enzymes last to minimize their exposure to the organic solvent.
5. The CYP concentration appears to be too high. I base this on the observation that the slopes of all the graphs for different CYP concentrations are about the same from 7.5 minutes onward. This suggests that the reactions have gone to completion and all that remains is a constant background rate that is not due to the activity of the enzyme.
6. The y-axis label should be relative fluorescence, not absorbance.
7. It might be helpful to include 0.01% Triton X-100 detergent in the assay buffer to keep the DBF and enzymes from sticking to the plate.
Adam,
Thank you so much for your suggestions. I really appreciate your gratefulness to advice me.
1. I will try removing NADP.
2. Yes, it was 538. It was type to write 438.
3. I will try DMSO. Let's see what happens.
4. Which two enzymes (G6P and CYP2C9 ??). So, you mean I should use three mixes. First goes in master mix, then substrate and then finally enzyme?
5. Ok. I will try 0.5, 0.25, 0.125, 0.05 mg/ml as well.
6. Yes, it was fluorescence (again a typo!!)
7. I will try add 0.01% detergent as well.
Please let me know your suggestion for point 4 and I will try the new assay.
Regards,
Pratik
This is how I would do it, just my personal preference.
All solutions would be made up to the necessary dilution in the assay buffer.
1. Add inhibitor at 3X final concentration.
2. Add DBF, G6P, NADPH mixture at 3X final concentration.
3. Initiate with G6PDH and CYP mixture at 3X final concentration. Alternatively, you could include the G6PDH with DBF, G6P and NADPH.
DBF should be protected from light as much as possible because fluorescein-related compounds are prone to photobleaching.
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