Biomedical Engineering Reference
In-Depth Information
3.3.5 Determination of Void Volume V
0
and Total Volume V
t
A
PBS
Solutions/Reagents
/
B2mg
ml dextran blue (mean M
r
>
2000 kD) in PBS
/
C1mg
ml dinitrophenylalanine in PBS
Equilibrate a packed GPC column, e.g., Superdex 200, 0.9 cm diam-
eter, 50 cm bed height, 32 ml bed volume, with 350 ml PBS. Adjust
the flow rate to 30 cm
/
/
min).
Apply 0.5 ml of Soln. B as described in part 3.3.3 and continue
the elution with Soln. A. Monitor the elution either continuously
by UV measuring at 280 nm or by collecting 1-ml fractions, which
are measured at 280 nm. Plot the optical density against elution
volume (cf. Fig. 3.4). The void volume V
0
is the volume at maximal
optical density.
For determination of the total volume V
t
use Soln. C instead of
Soln. B.
Calibrate a GPC column for molar weight determination anal-
ogously. Use a set of defined proteins or polypeptides as standards
(see inset in Fig. 3.4). If the mass differences allow a complete
separation, the proteins can be applied as a mixture.
For characterization of a GPC medium other than defined
macromolecules as dextrans, polypeptides, polystyrene sulfates,
or colloidal gold may be used. For these standards the eluent is
ddH
2
O. These macromolecules are suitable for molar mass deter-
mination of proteins with restrictions only.
h (0.3 ml
References
Amersham Biosciences (2002) Gel filtration: principles and methods
(www.chromatography.amershambiosiences.com)
Stellwagen E (1990) In: Deutscher MP (ed.) Guide to protein purification.
Academic Press, San Diego, p 317
3.3.6 Removing of Unbound Biotin After Conjugation by Gel
Filtration (“Desalting”)
Instead of dialysis, small molecules, such as salts or organochemi-
cal substances, can be separated quickly from macromolecules by
gel filtration. Buffer exchange is also possible this way. The disad-
vantage is a dilution of the sample.
The following example illustrates the fast removal of unbound
biotin after conjugation to a protein (Table 3.2).
/
/
A2mg
ml dextran blue (mean M
r
>
2000 kD), 0.2 mg
ml Pon-
Solutions/Reagents
ceauSinPBS
B
0.02% sodium azide (w/v) or 1% Neolone (v/v) in PBS
PBS
