Preparation polyacrylamides

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. 

Both agarose and polyacrylamide gels are solid but porous matrices, which look and feel like clear jelly (Jell-0). These gels are prepared in different ways but both take the form of a slab (or alternatively column), cast like a jelly in a mould. While agarose gels are relatively easy to prepare, polyacrylamide gels are formed by complex polymerization and chemical cross-linking, and as such are usually purchased pre-cast. 

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. 

In general, the elution of proteins above 100 kDa from polyacrylamide gels always presents considerable problems. Another of the serious limitations of elution from gels is owing to the elastic nature of preparative polyacrylamide gels. The precise excision of a protein band from a complex mixture is difficult and the slice may contain portions of other protein bands located close to the band of interest. To overcome some of the limitations of elution from gels, Parekh et al. (6) and Anderson (7) attempted to elute proteins... 

Hjerten and his research group [41] introduced monolithic columns based on acrylamides as chromatographic separation media in the late 1980s. Xie et al. [42] prepared rigid porous polyacrylamide-co-butylmethacrylate-co-A,A -methylene-bis-acrylamide monolithic column for hydrophobic interaction chromatography. They also prepared polyacrylamide-co-At, M-methylene-bis-acrylamide monolithic rods and smdied the effect of polymerization conditions on rods morphology. 

To start a run, the reactor was first filled with a previously prepared polyacrylamide solution more or less of the same description as the product desired. Nitrogen gas was turned on and the contents of the reactor were heated to the desired temperature. The two feed solutions were then pumped into the reactor. The temperature of the reactor contents would drop by 5° to 10° C. as the cold... 

Recovery of compounds by electrophoresis, continued until the sample migrates off the end of the supporting medium, can also be achieved for some forms of electrophoretogram, for example, preparative polyacrylamide rods, although this requires a special attachment at the base of the column to bleed off the sample as it emerges. 

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). 

Bottom panel Recovery of protease activity from preparative polyacrylamide gel of poliovirus proteins. 

Whenever possible, commercially prepared polyacrylamide gels were used. This avoids exposure to toxic chemicals and at the same time enhances reproducibility... 

Those cationic polymers in use for water treatment are nearly all based upon quatemized aminoesters or amino amides. Few non-ionic polyelectrolytes are used in water treatment and tme non-ionic types are difficult to prepare. Polyacrylamides containing less than 1% hydrolysable groups are available. High molecular weight poly(ethyleneoxides) are used in flotation processes. 

Table 1. Monomers utifizcd for preparing polyacrylamide gels...

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.

Streptomyces granaticolor after induction with different inducers arbutin (lane 1), methyl-3-D-glucosidase (lane 2), cellobiose (lane 3), salicin (lane 4), cellobiose + glycerol (lanes 5,6) -the protein band which exhibit the 3-D-glucosidase activity was partially purified by elution from preparative polyacrylamide gel. White bands at the right side of each lane indicate the position of -D-glucosidase activity. 

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. 

To improve the compatibility between SNPs and hydrophilic polymers, modification of SNPs was performed before mixing with the polymers or monomers (Yang et al., 2013b). Aluminum-modified colloidal sihca was used to prepare polyacrylamide (PAM)/silica NC gels with enhanced mechanical performance by in situ polymerization of AM in the presence of aluminum-modified silica. The hydrogel showed up to 15 times in compression stress compared with PAM hydrogel because... 

The preparative polyacrylamide slab gel electrophoresis is also effective for the purification of amino acid dehydrogenases due to the high separation ability and the easy... 

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