Gels Mimicking Antibodies in Their Selective Recognition of Proteins and Its Potential Use for Protein Crystallization - Molecules: Nucleation, Aggregation and Crystallization - page 18

The degree of selectivity of the adsorption

Horse myoglobin (3 mg) was dissolved in 1 mL of 10 mM sodium phos-
phate, pH 6.2. Acrylamide (57 mg), N,N ′ -methylenebisacrylamide (2.5

mg) and 10 L of a 10% aqueous solution of ammonium persulfate were

added. Following deaeration for one minute the solution was supple-
mented with 5

µ

L of a 5% aqueous solution of TEMED. The polymeriza-
tion proceeded overnight. The gel formed was pressed through a 60-mesh
net and packed into a Pasteur pipette (i.d.

µ

4 cm) with
glass wool at the outlet. The column was washed overnight with a 10%
(v/v) solution of acetic acid containing 10% (w/v) sodium dodecyl sulfate
to remove the horse myoglobin and was then equilibrated with 10 mM
sodium phosphate, pH 6.2. This column is denoted “horse myoglobin
column.” Another column was prepared in the same way, but in the
absence of horse myoglobin (“blank column”). Horse myoglobin, whale
myoglobin, ribonuclease and cytochrome c were dissolved in 10 mM
sodium phosphate, pH 6.2. The final concentration of each protein was
1 mg/mL. A 10

= 5 mm; bed height =

L volume of this sample was applied on each of the two
columns. Non-adsorbed proteins were eluted with the phosphate buffer
used for equilibration of the columns and analyzed in a cation exchanger.
Detection was done at 220 nm. The chromatogram in Fig. 4(a) was
obtained from the “blank column” and that in Fig. 4(b) from the “horse
myoglobin column.” Horse myoglobin was adsorbed only on the latter
column, and whale myoglobin on neither of the two columns. Figure 5
shows that the 3-D structures of these proteins are similar.

µ

Selective bed with higher flow rate

The polyacrylamide beds described above had the composition T6, C5.
The beds described previously had a similar composition (Liao et al .,
1996). From molecular-sieve chromatography experiments it is known
that such gels give a relatively low flow rate (Hjertén, 1962). Since this can
be improved by using denser, i.e. more rigid, gels, we prepared a bed with
the composition T20, C3. However, the selective adsorption of proteins