Biology Reference
In-Depth Information
6. DEAE purifi cation can then be performed to eliminate
contaminants that are often not visible by SDS-PAGE (i.e.,
nucleic acids).
7. Quality control can be performed by negative staining electron
transmission microscopy (
see
Fig.
2
).
1. Use dodecahedron at 1 mg/mL in HBS buffer (
see
Note 2
).
2. Dissolve the dyes in DMSO to get a working concentration of
10 mg/mL.
3. Add 10
3.3 Dodecahedron
Labeling and Cellular
Traffi cking
L of dye to 1 mL of dodecahedron solution, mix
immediately, and store in the dark at room temperature for 2 h
(
see
Note 6
).
4. Dialyze three times against 500 mL HBS buffer using a high
molecular weight cut-off MWCO 100 kDa (
see
Note 3
).
5.
For fi xed cells
: Incubate the coverslip with 1
μ
μ
g of labeled Dd in
L of medium without serum for the desired period of time.
Fix the cells with PFA 2 % for 20 min at 37 °C, then permeabi-
lize for 3 min with PBS—0.1 % triton ×100 (
see
Notes 7
and
8
).
6. Coverslips are mounted onto the glass slide using a 50 % glyc-
erol/50 % PBS drops and sealed with nail polish.
7.
For live-cell experiments
: Seed Iwaki dishes (0.17 mm glass
thick) to get 60 % confl uency. Add labeled dodecahedron (2
50
μ
μ
g
in 200
L) for 15 min at 37 °C. Remove the sample and add
pre-warmed medium. Observation is done in the thermostated
chamber of the microscope (
see
Notes 7
and
9
).
μ
For the general procedure:
3.4 Surface
Plasmon Resonance
1. Insert CM5 or CM4 (higher sensitivity) Sensor Chip.
2. Run with HBS or HBS supplemented with 2 mM CaCl
2
if
calcium is required for the interaction (e.g., cadherins, integ-
rins, etc.).
3. Activate two fl ow cells with EDC-NHS according to manufac-
turer instructions. 10 minutes contact is recommended (e.g.,
50
L/min).
4. Short cut the control Flowcell by working only on the “assay”
reference Flowcell (e.g., stop Flowcell 1, work only on
Flowcell 2).
5. Inject ligands at 10
μ
L injection at fl owrate 5
μ
μ
g/mL diluted in 10 mM acetate buffer
L/min for 10 min. Reinject at the appropriate
concentration if immobilization has not reached the expected
RU (e.g., 2,000-5,000 RU for ligand with a MW of 60 kDa,
200-500 RU for ligand with 6 kDa MW).
6. Block the chip with a 10-min injection of 1 M Ethanolamine
solution on both fl ow cells.
pH 4.5 at 5
μ
