Biomedical Engineering Reference
In-Depth Information
Fig. 1
Purifi cation of the
trans
-Golgi-localized tethering protein GCC185.
Coomassie-stained SDS-polyacrylamide gel of lysate input (
lane 1
), high-salt
washes (
lanes 2
and
3
), purifi ed GFP-FLAG-GCC185 full length (
lanes 4
and
5
),
and N-terminal half (1-889) before (
lane 6
) and after gel fi ltration on Sepharose
CL-4B (
lane 7
)
assessed by Coomassie Blue staining of SDS-PAGE gels. The
smaller, N-terminal half of GCC185 has higher yield than the
full-length protein (Fig.
1
). The next step is designed to remove
3× FLAG peptide and residual contaminants by size exclusion.
18. Prepare a 12.5 ml Sepharose CL-4B column (
see
Note 14
) and
equilibrate the column with two column volumes (25 ml) of
storage buffer (
see
Note 15
).
19. Carefully load 300
l of the FLAG peptide eluent without dis-
turbing the resin and allow the sample to enter the packed bed
completely (
see
Note 16
). Discard the fl ow-through.
20. Place tubes for sample collection under the column.
21. Elute with a total of one column volume (12.5 ml) of storage
buffer and collect 0.5 ml fractions.
22. Perform SDS-PAGE to detect fractions containing the target
protein and to evaluate purity (
see
Note 17
). The N-terminal
half of GCC185 elutes at 6.0-6.5 ml from a 12.5 ml Sepharose
CL-4B column with high purity (Fig.
1
).
ʼ
3.2 Monitoring
Tethering Function by
Immunofl uorescence
Microscopic Staining
In this type of functional rescue experiment, cells are depleted of
the tether with siRNA for a total of 72 h. Rescue constructs are
transfected into cells 24 h after initial siRNA treatment (
see
Note 18
)
so that the time permitted for rescue is 48 h (
see
Note 19
) overall.
1. HeLa cells are cultured in DMEM supplemented with 7.5 %
fetal bovine serum, 100 U/ml penicillin, and 100
g/ml
streptomycin in a 37 °C tissue culture incubator supplied with
5 % CO
2
.
ʼ
