Thanks for your reply.
I am sorry my post was not clear enough.
My tomato leaves were vacuum infilitrated with MG115 (a proteasome inhibitor), water, and FB1 (a fungal toxin). Our lab focuses on disease resistance and plant defense responses. The treatments serve as stress. We wanted to test the relative activity of certain enzymes under those stresses. The two enzymes are B-1,3-glucanase and glutamine synthetase.
I first extracted total protein by adding 1ml of 200mM Tris-HCl buffer (0.25mM EDTA, 5mM DTT, and 1mM PMSF) pH 8.0 to 0.5g of frozen tomato leaves and grinded them using a mortar and pestle. I made sure the mortar and pestle were cooled down with liquid nitrogen. The homogenate was centrifuged at 12000g at 4C for 20min.
The concentration of my protein samples was determined using the standard Bradford method.
The reaction mixture consisted of 0.4ml of citric-acid phosphate buffer (pH5.6) containing 1mg/ml laminarin and 0.1ml of enzyme solution (this is my total protein samples, keep in mind that after determining their concentrations, I diluted all to the same final concentration). After 15 min incubation at 37C, 0.5ml of the alkaline copper reagent was added and the mixture was heated at 100C for 10 min. After cooling on ice, 0.5ml of the arsenomolybdate reagent was added, followed by 3.0ml of water after the development of the blue color. The absorbance was measured at 660nm.
Then to measure the relative activity, I set the water treatment as (100%) and I measured the relative activity of the other treatments with respect to water.
Ex. if water-treatment absorbance is 0.56 and the FB1 treatment is 0.97, I divided 0.97/0.56*100= 173.21%
Glutamine Synthetase (GS)
We adapted a method for wheat, even though there are publications with tomato GS methods.
GS activity was measured using the
synthetase assay based on the method described by Lea
et al. (1990) and optimized for wheat leaves as follows.
100uL of crude leaf extract (the same protein samples were used as the ones used in glucanase assay above)was added to 380uL of assay
mix which consisted of 100 mM TEA, 80 mM glutamate,
6 mM hydroxylamine HCl, 20 mM MgSO4, 4 mM EDTA
at pH 7.6. The reaction was started by the addition of 20 uLof 0.2 M ATP at pH 7.6. After 10 min of incubation at
30C, the reaction was stopped by the addition of 500 uL of
ferric chloride reagent (0.24 M TCA, 0.1 M ferric chloride,
1.0 M HCl). Samples were then centrifuged at
10,000 g for 5 min and absorbance read at 505 nm.
We also looked at the relative activity of the enzyme for the different treaments and it was done the same way as that in the glucanase assay above.
I am sorry Bob as this is really long but I wanted to make sure that I am very clear on this. Please if you have more questions or not sure about anything, let me know