Biology Reference
In-Depth Information
12.
Following sonication, centrifuge the adipocyte extracts at 5000
g
for
10 min at 4
C. After collecting the supernatants, determine and
normalize protein concentration among different samples.
Enzyme assay
1.
Begin the assay by combining 100
m
L of adipocyte extract with 100
m
L
of substrate solution in a 15-mL conical tube. Thoroughly mix by
pipetting. Incubate the reaction mixture at 37
C for 1 h.
Note
: As with any enzyme reaction, timing of the substrate addition and
length of the incubation are critical in producing reliable results. Be sure to
accurately time the reactions.
2.
As the reaction is proceeding, prepare the MCH/STOP buffer by
thoroughly mixing 200 mL of methanol, 180 mL of chloroform, and
140 mL of heptane (10:9:7).
3.
At the end of the reaction period, add 3.25 mL of MCH/STOP buffer
and 1.0 mL of boric acid solution (described in
Section 1.3
) to each
sample. Mix thoroughly by vortexing for 15 s. Again please ensure that
timing is correct.
4.
Spin all tubes at 800
g
for 15 min at room temperature.
5.
Transfer 1.0 mL of the clear supernatant to a scintillation vial containing
10 mL of scintillation (counting) cocktail. Be careful to avoid the
lower phase.
6.
Determine radioactivity using a scintillation counter.
2.
Lipolysis assay
1.
Adipocytes in six-well plates are washed twice with DPBS and then incubated
in 2 mL/well of serum-free DMEM/0.5% BSA.
2.
After 3 h of serum starvation, the media is replaced with 2 mL/well of
serum-free, phenol red-free DMEM/2% BSA either containing or not
containing 10
M of isoproterenol (ISO). Serum starvation ensures
quenching of cAMP levels and 2% BSA in DMEM is important for capturing
any FFAs released from the cells into the media.
3.
Without disturbing the cells, remove 100
m
L of the media at 0.5, 1, 2, or 3 h
time points following ISO treatment. Aliquots of the media can be stored
at
m
20
C if FFA or glycerol content is not measured immediately.
4.
At the conclusion of the media collection, wash the cells with ice-cold DPBS
and proceed to lysis/extraction of proteins for immunoblotting analysis.
5.
Determine the glycerol or FFA content released into the media using
commercially available reagent kits as described in
Section 1.2
.
6.2.4
Case study—evaluating the roles of perilipin 1 and CGI-58
in ATGL-mediated lipolysis
The goal of this study was to examine the respective impact of perilipin 1 and CGI-58
on ATGL action in differentiated 3T3-L1 adipocytes (
Yang et al., 2013
). To this end,
we first subtracted endogenous perilipin 1 or CGI-58 through siRNA knockdown and
