Gels Mimicking Antibodies in Their Selective Recognition of Proteins and Its Potential Use for Protein Crystallization - Molecules: Nucleation, Aggregation and Crystallization - page 13

proteins, only the protein present during the polymerization will become
adsorbed. In this paper we present further studies of selective beds and
discuss the mechanism of selective recognition.

Experimental Details

Materials

Ribonuclease, lysozyme, cytochrome c , and myoglobin from horse and
whale were obtained from Sigma (St. Louis, MD, USA). We obtained
growth hormone as a gift from Professor Paul Roos, Department of
Biochemistry, Uppsala University and human serum albumin from
Dr Lars-Olov Andersson, KABI (Stockholm, Sweden). Hemoglobin was
prepared from human blood. Acrylamide, N,N ′ -methylenebisacrylamide,

ammonium persulfate, N,N,N -tetramethylethylenediamine (TEMED),

sodium dodecyl sulfate (SDS), and a cation exchanger (CB-S, i.d. 7 mm,
length 40 mm) were from Bio-Rad Laboratories (Hercules, CA, USA).

′ ,N ′

Preparation of a ribonuclease-specific gel

Acrylamide (5.82 mg), N,N -methylenebisacrylamide (1.8 mg) and

ribonuclease (3 mg) were dissolved in 1 mL of 0.01 M sodium phosphate,
pH 7.0. Following the addition of 20

′

µ L of a 10% (w/v) solution of

L of a 5% (v/v) TEMED solu-
tion was added. The polymerization proceeded for 30 minutes, producing
a gel with the total concentration ( T )

ammonium persulfate and deaeration, 20 µ

= 6% (w/v) and the crosslinking con-

centration ( C ) 3% (w/w), [for definitions of T and C see Hjertén (1962)].

The formed gel was then pressed through a 60-mesh net to produce gran-
ules, which were packed into a Pasteur pipette with glass wool in the con-
striction as support for the gel particles. The Pasteur pipette had an i.d. of
5 mm and the bed height was 4.5 cm. The granules were washed with
0.8 mL of a solution of Savinase (a proteinase obtained from Novo
Nordisk A/S , Denmark) to remove ribonuclease and equilibrated with

=