Biology Reference
In-Depth Information
2.
Add 0.1 mg of FITC to 1 mL of labeling buffer. Immediately add the cold FITC
solution dropwise until the final molar ratio of FITC to protein is 5:1 and incu-
bate overnight at 4
°
C with gentle rotation.
3.
Remove unconjugated FITC by exhaustive dialysis against deionized water at
4
°
C (
see
Note 2
) and freeze-dry to constant mass.
3.1.2. Hyaluronan Labeling
1.
C with gentle
stirring. Add 0.2 mL of CNBr solution (10 mg/ml in H
2
O, make up just before
use,
caution—toxic and hazardous
) and leave for 5 min (
see
Note 3
).
Dissolve 2 mg of HA in 1 mL of deionized water for 48 h at 4
°
2.
Isolate activated HA using 2 x Hi-Trap columns (Pharmacia, Uppsala, Sweden)
in series, elute with 0.2
M
borate buffer and collect 1 mL fractions. Add 2 mg of
fluoresceinamine (FA) to the activated GAG (fractions 4 - 6) and incubate over-
night at 4
°
C with gentle rotation.
3.
Remove salt and free FA by ultrafiltration at 4
°
C (Centriplus, Amicon, centri-
fuge 3000
g
, 10
washes with 10 mL H
2
O) and freeze-dry to constant mass.
Alternatively, free FA and salt may be removed by exhaustive dialysis, although
this takes considerably longer, especially for viscous hyaluronan solutions.
×
3.1.3. Preparation of Samples for Confocal FRAP Analysis
Fluoresceine concentration in final solutions for analysis should be kept
below 1 m
M
to avoid fluorescence self quenching effects. For solution studies
below 10 m
M
ionic strength, or below pH 6.0, the fluorescence signal is greatly
reduced, thus producing noisy data at low concentrations. More heavily labeled
material may then be necessary
(20)
.
1.
Before using labeled samples, it is advisable to check for the presence of
unconjugated fluorescein. Load 0.2 mg sample in 0.5 mL on a Hi-Trap column,
elute with PBS, and take 1-mL fractions. No fluorescence should be evident after
fraction 4 (
see
Note 4
).
2.
Make up solutions of labeled or unlabeled matrix macromolecules by weight and
equilibrate at 4
C for 48 h. In tracer studies, add FITC-labeled globular protein
probes (e.g., FITC-bovine serum albumin and FITC-soy bean trypsin inhibitor),
or polymer probes (e.g., FITC-dextrans) at <0.1 mg/ml and equilibrate for a fur-
ther 24 h at 4
°
°
C.
3.
L of sample onto a cavity slide and seal under a 20-mm diameter circu-
lar coverslip using nail varnish. Allow 10 min to dry (
Fig. 3
).
4. Place sealed cavity slide on thermally regulated microscope stage and allow
15 min to reach thermal equilibrium. Care should be taken to avoid condensation
in low temperature studies.
Pipet 30
µ
5.
Adjust the microscope stage height so the objective focal plane is midway
between the coverslip and the lower interface between the solution and the slide,
this is usually ~120
µ
m below the coverslip.
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