| GDP-ase Assay【Yale University】 | | 点击： 作者：51protocol收集 来源： 时间： 2007-03-18 本站论坛 |
|  | Use fresh tubes, 10 X 75 mm. Reaction mix is:
Stock
0.2M imidazole, pH 7.4 10 ul
0.1M CaCl2 2 ul
1% Triton X-100 10 ul
0.07M GDP 10 ul
the above can be mixed and
added together
H2O enzyme sample 68 ul
start reaction by adding the enzyme, vortex, and incubate at 37oC for
10 to 30 minutes. I incubate 30 min.
Stop reaction with 20 ul of 5% SDS. Vortex.
Add 880 ul of H2O.
Add 200 ul of acid molybdate (Sigma).
Add 50 ul of Fiske-Subbarow reducer (Sigma kit, prepare as bottle
describes). Vortex.
Read the A660 after 10-15 minutes.
Notes:
1. Run a blank without enzyme to blank machine. This is
essential since GDP is hydrolyzed by acid molybdate. Also
run a blank with enzyme but without substrate (no GDP)
for each sample and subtract out background.
2. More than 15-25 ul of 0.8M sorbitol buffer is inhibitory to
the color development. Sucrose solutions are not inhibitory.
I use 10 ul of column fractions (in sorbitol) and 30 ul of
sucrose gradient fractions.
3. CDP and ADP are not substrates for the enzyme and can be
used to correct for nonspecific reactions.
2
4. Procedure is similiar to Brandan and Fleisher.
5. The GDP-ase is Ca2 dependent. However, if using EGTA to
inhibit activity be very careful of the final pH of the sample.
Increasing pH results in increased background and signal,
possibly by activating other enzymes present in sample.
6. Do duplicates of each sample.
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