I am performing an Acetylcholinesterase and Butyrylcholinesterase inhibitory potential of plant extract, while doing so, i have followed an standard protocol followed by Rhee et al, 2001, using the standard compound Galanthamine as a positive control.
Here, i am confused about the
1. Standard- Galanthamine -and its IC 50 value
2. Solubility of an standard
3. Control concentration and its value to be
4. What is sample blank ? is it required to have a sample blank and How?
5. Blank (Auto zero) with buffer/H2O/any other solvent.
6. Concentration of a sample to be use for analysis
Proper assay protocol of AChE and BuChE is very crucial for me as a bud researcher, i am stuck in same assay for years , please help me to understand clearly i need your kind help to perform my assay.
The IC50s depend on what species the enzymes come from and the concentration of the substrate you are using, as well as the other experimental conditions such as pH and salts. To measure the IC50s in your own setup, prepare a set of serial dilutions of the inhibitor and measure the % inhibition at each concentration. For example, you could start at 100 micromolar, and prepare 2-fold serial dilutions down to about 0.098 micromolar (11 concentrations plus zero). That should cover the necessary range for galanthamine.
The solubility of galanthamine (or galantamine) hydrobromide in water is only about 1 mg/ml, according to the Sigma-Aldrich catalog, or about 3.5 millimolar, so it should not be a problem to test it at 100 micromolar.
In order to use galanthamine as a 100% inhibition control, you will need a concentration about 50-100 times higher than the IC50.
The purpose of the blank is to be able to subtract the absorbance coming from everything except the product of the reaction. It would be a good idea to have a blank for each inhibitor/sample concentration, at least at first. The blank should probably include the substrate but leave out the enzyme, assuming the substrate contributes some background and the enzyme doesn't. However, if the enzyme contributes background in the assay and the substrate doesn't, you should leave out the substrate instead. It is unnecessary to blank with water or buffer alone. The blank should have as many of the components of the reaction as possible, except the product.
As with the control inhibitor, a wide range of plant extract concentrations should be tested, with blanks for each concentration.
Adam B Shapiro Sir, thank you so much for your kind suggestion i am so glad for your reply on my question above again sir could you please tell me more about the control to be used and its constitution in details.
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