Biomedical Engineering Reference
In-Depth Information
Fig. 3.4.
Determination of V
0
and V
t
by gel permeation chromatography.
Dashed line:
dextran
blue (defines void volume V
0
);
dotted line:
DNP-alanine (defines total volume V
t
).
Inset:
plot of
a calibration curve for determination of molar mass
Swell 0.5 g Sephadex G-25 in 5 ml PBS for 3 h at RT. Then fill 1 ml
of the settled gel into an empty column (0.5- to 0.8-cm diameter)
and wash the bed with 20 ml PBS. Remove excess buffer from the
bed surface after washing.
Determine V
0
and V
t
by chromatography of 0.5 ml of Soln. A.
The blue fractions represent that volume which will contain the
biotinylated conjugate, and the red fractions will give the waste
(unbound biotin). After chromatography, equilibrate the column
again with PBS.
Dilute the mixture of biotinylation to 0.5 ml with PBS. Carefully
pour the solution onto the surface of the bed, allow the mixture to
