Polyacrylamide columns

Purification of Monoclonal Antibody. Immunoglobulins were precipitated from the pooled ascites by addition of an equal volume of saturated ammonium sulfate [50% (NH4)2S04]. The precipitate was collected by centrifugation (20 min 10,240 X g), dissolved in 0.01 M sodium phosphate (pH 6.8), and reprecipitated. After the second ammonium sulfate precipitation, the pellet was dissolved in a minimum volume of 0.01 M sodium phosphate (pH 6.8) and centrifuged for 10 min at 10,600 X g). The resulting supemate was applied to a P6G, gel filtration polyacrylamid, column (Bio-gel Biorad, Rockville Center, NY 1.5 X 40 cm). Fractions containing protein were pooled and applied to a hydroxyapatite column that had been equilibrated with 0.01 M sodium phosphate (pH 6.8). Proteins were eluted with a linear gradient of 0.01 to 0.3 M sodium phosphate. 

All these studies on chromatographic size exclusion separations on neutral separation media and selectivity of transportation of ions through membranes were carried out with very dilute electrolyte solutions. Only Rona and Schmuckler [135] examined Dead Sea concentrated brine on a Bio-Gel P-2 (crosshnked polyacrylamide) column and obtained a Hthium-enriched fraction free of calcium and magnesium. Bio-Gel P, however, is known to retain cations and probably enters hydrogen-bond interactions between the anions and the amide hydrogen. The elution order of chlorides was thus different from that expected for pure SEC, namely, K, Na, Li, Mg, and Ca, all, however, emerging before the hold-up (dead) volume of the column. 

Sudor, J., Foret, F., and BoCek, P., Pressure refilled polyacrylamide columns for the separation of oligonucleotides by capillary electrophoresis, /ecfn3p/jon sw, 12, 1056, 1991. 

Several procedures have been developed for the isolation of ADP-rihosylated nuclear protein. In one described by Hayaishi and coworkers (162) ADP-ribosylated nuclear proteins were separated by affinity chromatography in 6 M guanidine- HCl on a dihydroxyhoryl polyacrylamide column (162). A very specific interaction of the borate residue with the cis diol portion of ribose rings of the ADP-ribose permits the selective isolation of ADP-ribosylated proteins. Applying this technique to rat liver nuclear proteins, they observed a distribution of ADP-ribosylation between histone and nonhistone proteins, with histone H2B (67%) and Hi (33%) being preferentially modified. 

FIGURE 8.12 Effect of pore diameter on SEC of standards (nondenaturin > mobile phase). Nondenaturing" refers to the effect on the stationary phase. Most iarge proteins were in fact denatured by this mobile phase (which was optimized for use with peptides, not proteins). Accordingly, it was necessary to use polyacrylamide to demonstrate the approximate range and position of Vo under these conditions. The polyacryiamide standards both eiuted at V with the 300-A coiumn (not shown). Columns and flow rate Same as in Fig. 8.11. Mobile phase Same as in Fig. 8.1. Sample key (B) Ovalbumin (43,000 Da) 0) polyacrylamide (1,000,000 Da) (K) polyacrylamide (400,000 IDa) (L) low molecular weight impurity in the polyacrylamide standards. Other samples as in Fig. 8.11. 

FIGURE 9.23 Analysis of ultrahigh poly(acrylamjde). MW 48 million by analytical ultracentrifugation. Eluent 0.1 M Na2SO<. Flow rate 0.3 ml/min. Columns PSS Suprema 30000, 20 /tim, 8 x 300 mm. Oven temp 30°C. Detector Rl. Standards PSS polyacrylamide standards. 

The larger macromolecules can be separated using larger particle size columns. However, the flow rate should be watched carefully. As the effective hydrodynamic size of the macromolecules may be reduced due to the deformation by shear (23). Figure 22.8 shows that the effective hydrodynamic size of a 12-15 X 10 MW polyacrylamide sample will not reach its maximum, or the size without shear, unless the flow rate is reduced to 0.01 ml/min. A... 

Figure 9.3 Schematic illustration of the electrophoretic transfer of proteins in the chromatophoresis process. After being eluted from the HPLC column, the proteins were reduced with /3-mercaptoethanol in the protein reaction system (PRS), and then deposited onto the polyacrylamide gradient gel. (PRC, protein reaction cocktail). Reprinted from Journal of Chromatography, 443, W. G. Button et al., Separation of proteins by reversed-phase Mgh-performance liquid cliromatography , pp 363-379, copyright 1988, with permission from Elsevier Science.

Fig. 10. HPLC of proteins (commercial samples) on the /V-butyl polyacrylamide coated silica gel column. Sample 20 pi of 5-15 mg/ml protein solution in buffer A. Buffer A 10% methanol, 0.2 mol/1 ammonium acetate, pH 4.5. Buffer B methanol. Gradient 50-min linear, 0-100% B. Flow rate 0.8 ml/min. Peaks (/) — lysozym, (2,3) — insulin, (4,5) — myoglobin [57]...

Figure 5. Analytical isoelectric focusing. Ultrathin layers (0.4 nun) of polyacrylamide with ampholytes pH 2-11 were used. Samples of 10 pg of protein in 10 pi of 1 % glycine were applied. A.- Silver staining. B.- Stain for activity on overlays containing pectin in tris/HCl buffer at pH 8.0 with CaClj M.- Broad pi Calibration Kit protein (Pharmacia), samples of 5 pg of protein were applied. 1.-Ammonium sulphate precipitated proteins from cultures on pectin. 2.- Fractions with PNL activity eluted from the Superdex 75HR1030 column. 3.- Purified PNL.

Figure 2. Molecular weight calibration curves for nonionic polyacrylamide for a single column (4 ft X Vs in. i.d.) containing 2000 A CPG-10 (200/400 mesh) packing with aqueous salt solutions as mobile phase.

Figures 6, 7 and 9 show calibration curves using two multi-column combinations and illustrate the degree of "optimization obtained in this system. The mobile phases for Figures 6 and 7 contained 0.025 g polyethylene oxide and ion exclusion and adsorption effects should therefore be largely eliminated. Figure 6 shows that reasonably good resolution can be obtained with a combination of five columns but does exhibit some loss of peak separation at the low cuid high MW ends. In Figure 7 the effect of adding a sixth column of small pore size is illustrated and it is seen that resolution at the low MW end is thereby somewhat improved. This calibration curve is effectively linear with a change of slope at 500,000 MW. It should provide a useful aqueous GPC system for MW and MWD determination of nonionic polyacrylamides.

Figure 7. Molecular weight calibration curve for nonionic polyacrylamides for a 6-column combination (each 4 ft X in, Ld.) with 3000 A, 3000 A, 2000 A, 1000 A, 729 A, and 500 A CPG-10 (200/400 mesh) packing.

Figure 8. Chromatograms for nonionic polyacrylamide standards and Standard C for the column combination shown in Figure 6.

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