Preparative polyacrylamide gel electrophoresi

The Rotofor has been incorporated into two-dimensional purification schemes based on the principles of 2-D PAGE. Fractions collected from the Rotofor were further purified by 2-D PAGE62 or by preparative polyacrylamide gel electrophoresis.63-65 Highly purified proteins were obtained by this scheme, even low-abundance proteins, to allow for multiple analyses and for use as antigens. 

Macfarlane, D.E. (1989) Two dimensional benzyldimethyl-n-hexadecylaimnonium chloride-sodium dodecyl sulfate preparative polyacrylamide gel electrophoresis a high capacity high resolution technique for the purification of proteins from complex mixtures. Anal. Biochem. 176,457-463. 

D-Mannanase (j3-D-mannosidases) purified from the germinated seeds of Trifolium repens by a procedure that included chromatography on hydroxyapatite, gel filtration on acrylamide/agarose (Ultragel 5/4) and preparative polyacrylamide gel electrophoresis. The final purification has been completely resolved into two ]3-D-mannanases (I and II) with distinct specificities (see p. 468). 

Preparative polyacrylamide gel electrophoresis. The gel is hollow cylinder-shaped with outer diameter 46 mm, inner diameter 20 mm, and 90 -100 mm in height. Both sides of the gel were cooled and electrophoresis was usually carried out at pH 8 with a constant current of 25 mA after loading with 50 to 70 mg of protein. After about 16 h electrophoresis, the gel was taken out from the apparatus (Toyo Filter Co., model CD 50), sliced into rings 2.5 mm thick, broken by syringe passage. 

Fig. 1. Purification of cellulase and 3-glucosidase by Toyo-pearl TSK-HW55 column chromatography. The active fractions in D A Sephadex A-50 chromatography were charged after concentration. 31> 32, CII, cm, CIV and CV indicated in the figure were pooled and concentrated and further purified by preparative polyacrylamide gel electrophoresis.

Fig. 2. Purification of cellulase CIV by preparative polyacrylamide gel electrophoresis. The cellulase CIV fraction from HW-55 chromatography was charged. After electrophoresis at pH 8, the enzyme was eluted as described in Methods and the activities on CMC and PNPG in each fraction were determined.

Disc Electrophoresis. The preparative polyacrylamide gel electrophoresis was carried out according to the procedure of Stevens (1967). The instrument used was from Buchler Instruments, Inc., Fort Lee, N. J. 

Fig,3 Preparative polyacrylamide gel electrophoresis of partially purified urinary FSH (El61bis-C). 91 mg of protein applied to gel column 4 ml/tube. Recovery total protein 88.5%, FSH 95 3%. 

Table 3 Relative potencies and recoveries of the fractions obtained by preparative polyacrylamide gel electrophoresis of the partially purified FSH (El61bis-C)...

Interconversions based on a subunit model of the A, B, and S forms of isoenzymes of the j8-o-2-acetamido-2-deoxyhexosidase in human tissues have been accomplished by means of preparative polyacrylamide gel electrophoresis." It was concluded that the A, B, and S forms are composed of a heteropolymer containing a- and j8-chains, a jS-chain homopolymer, and an a-chain homopolymer, respectively. Separation of the isoenzymes of jS-D-2-acetamido-2-deoxyhexosidase in human tissues by electrophoresis on cellulose acetate membranes has been used in the diagnosis of GM -gangliosidosis." The relative abundances and properties of the three isoenzymes were determined, and the relevance of the results to the disease was discussed. 

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