Biomedical Engineering Reference
In-Depth Information
evenly spread on the day of the transfection (
see
Note 6
). Grow
cells at 37 °C and 5 % CO
2
in a 90 % humidifi ed incubator.
2. Prepare transfection solutions with the modifi ed manufactur-
er's protocol. For a 12-well plate, in a 1.5 mL microcentrifuge
tube, dilute 3
L of Opti-
MEM
®
, set aside, and let incubate for 5-10 min (
see
Note 7
).
3. In a separate tube, combine 1.6
ʼ
L of Lipofectamine™ 2000 in 50
ʼ
ʼ
g total DNA (multi-expression
COG DNA, 0.2
ʼ
g of each plasmid, and 0.8
ʼ
g of GFP-tagged
partner protein) with 50
ʼ
L of Opti-MEM
®
gently mixing the
solution (
see
Note 8
).
4. After 5-10 min, combine the diluted DNA with the diluted
Lipofectamine™ 2000 and incubate for 20 min.
5. After 20 min of incubation, add in dropwise manner the DNA-
Lipofectamine™ 2000 complexes to their corresponding wells.
Mix gently by rocking the plate back and forth (
see
Note 9
).
6. Incubate the cells for 8-12 h at 37 °C, 5 % CO
2
, and 90 %
humidity, then remove transfection solution and replace with
10 % FBS DMEM/F-12 growth media and allow cells to
recover.
7. 24 h after the transfection, proceed to harvesting for
co-immunoprecipitation.
3.3 Co-
immunoprecipitation
All steps of the immunoprecipitation are done at room tempera-
ture, with room temperature reagents.
1. HEK293T cells that have been transfected for 24 h with COG
multi-expression plasmids were collected by gentle resuspen-
sion in 1 mL of growth media (
see
Note 10
) and placed in a
1.7 mL microcentrifuge tube.
2. Centrifuge cells for 2 min at 1,000 ×
g
.
3. Remove growth media from tube using a vacuum and resus-
pend the cell pellet in 1 mL of 1× PBS (
see
Note 11
).
4. Centrifuge cells for 2 min at 1,000 ×
g
.
5. Remove PBS from the tube and resuspend the cell pellet in
0.5 mL of immunoprecipitation lysis buffer.
6. Incubate the cells in lysis buffer and allow lysis to proceed for
30 min.
7. Centrifuge the cell lysate for 10 min at 20,000 ×
g
.
8. Aliquot 50
L of lysate supernatant (S20) to a new 1.7 mL
microcentrifuge tube for analysis. Add 5
ʼ
ʼ
L of 6× sample buf-
fer and boil for 5 min.
9. Transfer the remaining S20 to a new microcentrifuge tube.
10. Add 25
L of 50 % washed GBP bead suspension (
see
Note 12
).
11. Incubate the beads and the lysate sample for 2 h on the rotator.
ʼ
