( ced in 14000 MW cutoff dial...)Preparative Nondenaturing Gel Electrophoresis to Purify NADP-Specific Glutamate Dehydrogenase From Chlorella, Rev B

ced in 14,000 MW cutoff dialysis tubing. The concentrated sample was dialyzed at 4C against 28.8 mM Tris, 192 mM glycine, 2 mM DTT (pH 8.4) for 30 min. The dialyzed enzyme preparation was clarified by centrifugation at 20,000 x g for 10 min and was combined with 3 ml of 40% sucrose and 1 ml of 0.02% Bromophenol Blue.

Preparative Nondenaturing Gel Electrophoresis
For preparative nondenaturing gel electrophoresis, a 3 cm high 7% acrylamide (28 acrylamide:0.735 bis-acrylamide, pH 8.8) resolving gel and a 2 cm high 2% acrylamide (1.6 acrylamide:0.4 bis-acrylamide, pH 6.6) stacking gel were cast in the 28 mm ID gel tube of the Model 491 prep cell. The resolving gel was polymerized in 374 mM Tris (pH 8.8) using 140 g/ml ammonium persulfate and 1.12 l/ml TEMED. The stacking gel was polymerized in 39 mM Tris (pH 6.6), 12.7% sucrose using 0.07% riboflavin-5-phosphate (Bio-Rad) and 0.365 l/ml TEMED. Stacking gel polymerization was achieved by exposure to two 5 W fluorescent lights for 45 min. Both gels were cooled as per the Model 491 prep cell instruction manual during the polymerization process. All acrylamide stock solutions were pretreated with AG 501-X8 mixed bed resin to remove any contaminating acrylic acid to prevent in vitro acylation of proteins during the electrophoresis process. The preparative gel was pre-electrophoresed for 10 min at 15 mA constant power in upper/lower gel electrophoresis buffer containing 28.8 mM Tris, 192 mM glycine (pH 8.4, 4C). The elution buffer reservoir was filled with elution buffer comprised of 28.8 mM Tris, 192 mM glycine, 2 mM DTT, and 0.1 mM NADP+ (pH 8.4, 4C). The entire Model 491 prep cell was cooled to 4C during the electrophoresis process
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