Biology Reference
In-Depth Information
3.
Preclear media samples and plasmin digests with nonimmune rabbit serum (This
step reduces background nonspecific bands, which may bind to the protein G
agarose or to other serum proteins). Do this by adding 5
L nonimmune rabbit
serum to each sample, then incubating for 30 min at room temperature on an end
over rotator.
µ
4.
Add 30
µ
L protein G agarose (Boehringer Mannheim) and incubate for 2 h at 4
°
C
on rotator.
5.
Microfuge at 14,000 rpm for 5 min then transfer the supernatant to a clean 1.5-mL
tube and discard the pellet. Add 5% BSA solution to the plasmin digests to give a
final concentration of 0.2% (This provides excess protein which may help reduce
nonspecific binding of radiolabeled proteins to the protein G agarose).
6.
Divide each sample and use one half for immunoprecipitation with the specific
antiserum (anti-LTBP1) and the other half for a nonimmune rabbit serum control
(this control is important as it indicates any bands that are nonspecific). Do this
by adding 5
L of the appropriate serum to each tube. Incubate 30 min at room
temperature on a rotator.
µ
7.
Add 30
µ
L protein G agarose and incubate for 2 h at 4
°
C on a rotator (
see
Note 7
).
8.
Microfuge at 16,000
g
for 5 min, discard the supernatant and wash the pellet as
follows; (a) 3 times in 1 mL wash buffer 1 (low salt), (b) 3 times in 1 mL wash
buffer 2 (high salt), and (c) one time in 1 mL PBS.
9.
Aspirate PBS from the pellet, then centrifuge again and aspirate more if possible
to leave the pellet as dry as possible.
10.
Add 30
µ
L 1X nonreducing sample buffer to pellet and store samples at -20
°
C
until ready to run SDS-PAGE.
11.
Boil samples for 10 min, then separate proteins on a 4-20% gradient SDS-PAGE
gel (load 25
L sample per lane). SDS-PAGE analysis is a standard procedure
performed in most laboratories and details of this technique will not be described
here. For a standard protocol and discussion of this technique please refer to
(17)
.
µ
12.
After performing SDS-PAGE, place the gel in a tray and fix with gel fixing solu-
tion 1 for 30 min at room temperature with gentle shaking.
13.
Change the solution to gel fixing solution 2 and incubate for a further 30 min at
room temperature with gentle shaking (
see
Note 8
).
14.
Pour off this solution and add sufficient En
3
Hance solution (Du Pont, NEN) to
cover the gel. Incubate for 1 h at room temperature with gentle shaking. This step
as well as the subsequent washes should be performed in a fume hood.
Pour off En
3
hance solution and wash the gel three times in distilled water for
20 min at room temperature.
15.
16.
Dry the gel on a gel drier and then expose dried gel using Kodak X-OMAT
AR5 film.
Figure 3
shows an example of how this method was used to examine release
of LTBP1 and latent TGF
β
from bone matrix by avian osteoclasts.
4. Notes
1.
The radiolabeling media contains one-tenth the cysteine content of normal culture
media. This is necessary because of the long labeling period used. If a shorter
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