Biology Reference
In-Depth Information
and quantified as the readout for the enzyme activity. Since lipolysis in adipocytes
produces both FFAs and glycerol, the lipolytic rates can be easily measured by de-
termining the per hour release of FFA and glycerol from the cells into the culture
media.
1.
TG hydrolase activity assay
Substrate preparation
1.
Prepare 10 mL of 20% FA-free BSA in 0.1 M potassium phosphate
buffer (pH 7.0).
Note
: The inclusion of BSA in the reaction mixtures serves the purpose
of capturing free oleic acid released from triolein hydrolysis in the water
phase.
2.
Prepare a 0.1 M boric acid solution in potassium bicarbonate and adjust
pH to 10.5.
Note
: Final concentration of potassium bicarbonate should also be 0.1 M.
3.
For 4 mL of substrate solution, add the following to a 12
75 mm glass
10
7
cpm of [
3
H]-triolein,
test tube: 5.9 mg of unlabeled triolein, 5
L of phospholipid solution (20 mg/mL phosphatidylcholine:
phosphatidylinositol, 3:1). Mix thoroughly by pipetting.
4.
Evaporate the solvents by placing the 10-mL glass test tube under a
constant nitrogen (N
2
) stream.
5.
Following complete evaporation, transfer the test tube to a desiccator
and dry under vacuum for an additional 15 min. It is imperative to
completely remove residual of any organic solvents.
6.
After complete drying, add 2 mL of 0.1 M potassium phosphate buffer
(pH 7.0) and sonicate for two cycles using a Misonix sonicator at an
output setting of 1-2. Each cycle should be 1 min in duration at room
temperature with a 1-min recovery interval on ice between cycles.
7.
Add an additional 1 mL of 0.1 M potassium phosphate buffer (pH 7.0)
and continue to sonicate for four cycles of 30 s each at room temperature,
with 30-s recovery intervals on ice between cycles. Output setting
remains at 1-2.
8.
Following sonication, add 1 mL of 20% FA-free BSA in 0.1 M
potassium phosphate buffer (pH 7.0) and mix thoroughly by pipetting.
Adipocyte extraction
9.
Prepare cell extraction buffer consisting of 0.25 M sucrose, 1 mM
EDTA, 1 mM DTT, and protease inhibitors (1 mini tablet/10 mL) as
desired in molecular biology grade water.
10.
Add 0.5 mL/well of extracts buffer to adipocytes in six-well plates and
scrape cells into a 1.5-mL microcentrifuge tube.
11.
Sonicate the cells using the Misonix sonicator for three cycles of 10 s
each at an output setting of 5, ensuring a recovery interval of at least
30 s between cycles.
Note
: Keep extracts on ice during and after sonication to maintain
protein integrity.
and 30
m
