We have tested this product with zebrafish brain and embryo lysates. A zebrafish mutant line sb55 (Behra et al., 2002) was used for the validation experiments as a control. In this mutant line homozygous mutants sb55-/- has abolished acetycholinesterase activity compared to wildype controls and homozygous mutation is lethal for embryos after 4 dpf due to paralysis. The measurement taken from homozygous mutants at 4dpf is approximately zero and there is a drastic difference between mutants and wildtypes at 4dpf in terms of acetylcholinesterase activity. In the adult fish mutation is not lethal for heterozygous mutants(sb55+/-) and heterozygous mutants have lower levels of brain acetylcholinesterase (Ninkovic et al., 2006). Our data is also confirming the previous observations by other groups working with zebrafish model and indicate that this product is giving quite accurate measurements with zebrafish brain and embryo samples.
Extraction Protocol: Brain tissues and embryos were homogenized in ice cold 100 ul of 25 mM Tris HCl pH:8. Then centrifuged at 13 000 rpm for 20 mins at 4°C. Collected supernatants were used for the assay.
Behra, M., Cousin, X., Bertrand, C., Vonesch, J.L., Biellmann, D., Chatonnet, A., and Strähle, U. (2002) Acetylcholinesterase is required for neuronal and muscular development in the zebrafish embryo. Nature Neuroscience. 5(2):111-118.
Ninkovic, J., Folchert, A., Makhankov, Y. V., Neuhauss, S. C., Sillaber, I., Straehle, U., & Bally‐Cuif, L. (2006). Genetic identification of AChE as a positive modulator of addiction to the psychostimulant D‐amphetamine in zebrafish. Journal of neurobiology, 66(5), 463-475.
Miss. Elif Karoğlu
Submitted May 10 2019
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