We started a protocol to study the anti-cholinesterase activity of some plants. After dilution of 0.2 mg in 1 ml of a Ph 8 buffer and further dilute 300 microliter in 2 ml as indicated in the protocol we got an absorption value very low (0.08) when the expected value in the protocol is 0.4-0.5.
would you give me the detailed protocol you use in your laboratory?
Please refer to our method that we have successfully used to screen anticholinesterase activities of plants/ essential oils/ compounds. To me, the spectrophotometric method works but, the bio-TLC is fun to do and results very convincing. In your description, few things I could observe which might have been problematic: (1) pH in 7.4-7.8 range and 8 may be a bit higher ? (2) Dissolve in terms of Units/ ml and not "mg" to make sure enough is there, (3) and if you use BTIC for BuChE then it would be more specific, (3) also the OD would be that low only for protein, so to get a higher OD, the "substrate "BTCI" needs to be added and incubated say 15-30 mins at 37 C temperature to see a color change at 415 nm (because justa protein solution would not give an OD at visible range !!), and (4) also make sure to use Quartz cuvettes to be on safer side although 415 nm is in visible range to avoid your spectro's insensitivity issues etc. Let me know if these help. Best wishes. Thanks, Biswa
Calibrate your spectrophotometer ; See if your 'commercial enzyme' is outdated or not; and calibrate the activity of the enzyme procured by using increasing substrate concentration to verify your enzyme works. (I would also use one substrate conc and increase the enzyme protein and see the activity).
If you are using an assay KIt, then see the expiry date.
All the best
You can try our spectrophotometric method (Karlsson et al., 1984) which is rather old but very reliable and simple to carry out. It is a modification of Augustinsson et al. (1978). It was used by us in our work on Fasciculins' purification in Karlsson´s lab BMC, Uppsala, Sweden.
Article Fasciculins, anticholinesterase toxins from the venom of the...
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