High resolution preparative isoelectric focusing in layers of granulated gels

Abstract

Discontinuous isoelectric focusing in layers of granulated gels is used to separate up to 1000 mg protein with a resolution of 0.01-0.015 PI. High resolution is achieved with (i) gel layers of reduced thickness (1 mm), (ii) high field strength in the final stage of focusing (100-200 V/cm), (iii) long separation distances, and (iv) high Volthour (Vh) products ensuring steady state conditions. We describe approaches to rapid focusing based on either short separation distances or extensive prefocusing with resultant short residence times of the separated substances in the gel. The optimum load capacity is 3-5 mg protein/ml gel-bed volume while 10 mg proteidml gel-bed volume are still well tolerated. At high loading, major componentsare seen in the gel as translucent zones which can be easily identified and harvested. Best results are obtained with Sephadex G-200 (Superfine) or Bio-Gel P-60 (minus 400 mesh). Protein visualization with a new printing technique employing cellulose acetate membranes requires only 2-3 min and is insensitive to protein overloading and adhering gel particles. A simple and efficient method is described for the electrophoretic removal of carrier ampholytes from the focused proteins. High resolution preparative isoelectric focusing should prove attractive for many applications due to its great flexibility with respect to sample size, gel volume, separation time, protein recovery, ease of operation and reasonable price.

Electrophoresis

“…Stable pH gradients can be achieved with ampholytes in such a stabilized medium with no disruption from convection. The lower portion of Figure 3 shows the result of isoelectric focusing of several proteins (7). The proteins were "fixed" and then stained with Ponceau S. The figure also shows the pattern that results from scanning a densitometer across this gel.…”

The structure of Artemia sp. (brine shrimp) haemoglobins. Purification of a structural unit to homogeneity

“…Preparative isoelectric focusing of the structural units was performed in a Sephadex G-75 (superfine) layer (21 cm x 25cm x 0.15cm), containing 4M-urea and 2% (v/v) Ampholines (pH range 4.0-6.5) as described by Radola (1973Radola ( , 1974 and Frey & Radola (1982).…”

“…A pure fraction containing a single structural unit type could only be obtained by isoelectric focusing (pH4.0-6.5) on a Sephadex G-75 (superfine) layer performed by the method of Radola (1973Radola ( , 1974 and Frey & Radola (1982) (Fig. 4).…”

“…Table 1. Analysis oJ the structural-unit fraction Fraction E was separated preparatively by isoelectric focusing on a Sephadex G-75 layer under native conditions (Frey & Radola, 1982), after which each fraction was analysed in a two-dimensional system under denaturing conditions as described by O'Farrell (1975 On SDS/polyacrylamide-gel electrophoresis (Laemmli, 1970), two bands with M, values of 15800+800 (n=6) and 17200+900 (n=6) respectively were obtained. E1 and E5 clearly contain single bands (Mr 15800 and 17200), whereas E2, E3 and E4 are double (Fig.…”