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Reagents:
(e) Acetylthiocholine iodide, 0.075 M (21.67mg/ml); it can be kept for 10-15 days at
4оC.
Procedure:
Reagents:
(e) Butyrylthiocholine iodide; 24 mg/ml; it can be kept for 10-15 days at 4 оC.
Procedure:
Reagents:
(d) NaCl, 2M
(n) SnCl2, 0.05% in 2.5 N H2SO4 (prepared fresh for each experiment)
Procedure:
• Enzyme sample was diluted and preincubated at 37оC for 5-10 minutes before
use.
• Volume was made up to 6.9ml with distilled water and placed in a 37оC water
bath for 5 minutes.
• For blanks, tubes were prepared containing no enzyme and containing enzyme
with no ATP mixture.
• After 10 minutes 0.5 ml of reagent i was added and placed in ice bath for 15
minutes.
• Removed 0.8 ml of supernatant to test tube in ice and added 0.2 ml of reagent k,
mixed well.
• Added 0.2 ml of n and mixed. Tubes were left for 15 minutes and absorbance
was read at 650 nm.
Direct colouring method described by Karnovsky and Roots (1964) was adapted
for the demonstration of AChE.
Incubation medium:
5 mg of substrate (i.e. acetylthiocholine iodide) was dissolved in 6.5 ml of 0.1M
phosphate buffer (pH = 6.0). Then following items were added in order while stirring-
Procedure:
Incubation medium:
Procedure:
Incubation medium:
ATP 0.125g % = 20 ml
Tris-maleate buffer (pH 7.2) 0.2M = 20 ml
Lead nitrate 2% = 3 ml
Magnesium sulfate 0.1M = 5 ml
Distilled water = 2 ml
Procedure:
• Sections were incubated at 37оC for 5-60 minutes in the incubating medium.
• Then the sections were treated with 1% yellow ammonium sulphide for one
minute.