Biology Reference
In-Depth Information
Table 12.1
Reagents
Dulbecco's phosphate-buffered saline (DPBS)
137 mM NaCl
2.7 mM KCl
1.5 mM KH
2
PO
4
8.1 mM Na
2
HPO
4
Adjust pH to 7.4
Lysis buffer
20 mM Tris-HCl pH7.4
1 mM EDTA
150 mM NaCl
1% TritonX-100
1mMNa
3
VO
4
Store at 4
C
Laemmli sample buffer
75 mM Tris-HCl
2% SDS
10% glycerol
3% 2-mercaptoethanol
0.003% bromophenol blue
Store at
20
C
BRET
2
buffer
DPBS containing
0.1 mg/ml CaCl
2
0.1 mg/ml MgCl
2
1 mg/ml
D
-glucose
Tyrode-HEPES solution
137 mM NaCl
2.7 mM KCl
1.0 mM MgCl
1.8 mM CaCl
2
10 mM HEPES
5.6 mM glucose
Adjust pH to 7.4
Store at 4
C
lysis buffer (
Table 12.1
). After rotating for 3 h at 4
C, the solution was centrifuged at
17,400
l/ml of
50% (v/v) Protein G Sepharose
™
4 Fast Flow (GE Healthcare, Piscataway, NJ) in
lysis buffer, followed by centrifugation at 17,400
g
for 20 min at 4
C, and the supernatant was precleared with 30
m
g
for 10 s to remove nonspeci-
fically bound proteins to Sepharose. Anti-myc 9E10 antibodies (10
g/ml) were
added into the supernatant and rotated for 1 h at 4
C. Fifty microliters per milliliter
of 50% (v/v) Protein G Sepharose
TM
4 Fast Flow was then added into the supernatant
and rotated for a further 2 h. The mixture was centrifuged, the resulting precipitate
was washed twice with 500
m
m
l of lysis buffer, and bound proteins were eluted by in-
cubation with 30
m
l of the Laemmli sample buffer (
Table 12.1
) at room temperature.
