Gels Mimicking Antibodies in Their Selective Recognition of Proteins and Its Potential Use for Protein Crystallization - Molecules: Nucleation, Aggregation and Crystallization - page 15

Preparation of a gel for specific adsorption
of human growth hormone

Human growth hormone (3 mg) was dissolved in 1 mL of 0.1 M sodium
phosphate, pH 6.2. Acrylamide (5.8 mg), N,N ′ -methylenebisacrylamide

(2.4 mg) and ammonium persulfate [10 L of a 10% (w/v) aqueous solution]

were added. The deaerated solution was supplemented with 10

µ

Lof a
5% (v/v) aqueous solution of TEMED. About 30 minutes following poly-
merization of the gel (which had the composition T6, C4; Hjertén, 1962) it
was granulated by pressing it through a 100-mesh net and packed into a
Pasteur pipette. To desorb the growth hormone, two column volumes of
sodium phosphate (0.1 M, pH 6.2) were passed through the column followed
by five column volumes of 10% (v/v) acetic acid containing 10% (w/v) SDS.
The column was washed with the buffer until the pH of the effluent
reached 6.2 and no precipitate of dodecyl sulfate could be detected upon
addition of potassium chloride (potassium dodecyl sulfate is only slightly
soluble in water). A 10

µ

L volume of the sample, a mixture of growth hor-
mone (5 mg/mL) and human serum albumin (2 mg/mL) in the phosphate
buffer, was added. The eluate was analyzed in an anion exchanger using a
5 min linear concentration gradient created by the equilibration buffer and
the same buffer containing 0.5 M sodium chloride (Fig. 2(c)). The eluate
from a blank column (prepared in the absence of growth hormone) was stud-
ied in the same ion exchanger (Fig. 2(b)), as was the original sample (Fig. 2(a)).
The chromatograms show that the growth hormone, but not albumin, had
adsorbed to the column prepared in the presence of the hormone.
For artificial gel antibodies against other proteins and for gel antibodies
against viruses and bacteria (see Hjertén, Liao (1998) and Takátsy et al .
(2006a; 2006b; 2007)). For other proteins, see Liao et al . (1996), Hjertén
et al . (1997) and Tong et al . (2001).

µ

Can a bed be designed with selectivity for more
than one protein?

Acrylamide (171 mg), N,N ′ -methylenebisacrylamide (9 mg), hemoglobin

[60 µ L of a 1% (w/v) aqueous solution], cytochrome c (5 mg) and