Disc gels

Hedrick, JL Smith, AJ, Size and Charge Isomer Separation and Estimation of Molecular Weights of Proteins by Disc Gel Electrophoresis, Archives of Biochemistry and Biophysics 126, 155, 1968. 

Chromatography and Electrophoresis of Antigens. Cotton dust (20-30 mg) was dissolved in water and passed through a small column (30 x 1.5 cm id) packed with 3 g of Sephadex G-75 that had been preswelled in water. Chromatograms were obtained with an LKB Uvicord ultraviolet monitor and a chopper bar recorder. Volumes of 5 ml each were collected in test tubes with an automatic fraction collector. Two components were detected at 250 nm. Samples of 10 mg were analyzed by disc gel electrophoresis according to procedures described by Davis (36), and Zacharius (37). 

The process of disc gel electrophoresis. A Before electrophoresis. B Movement of chloride, glycinate, and protein through the stacking gel. 

Disc gel electrophoresis yields excellent resolution and is the method of choice for analysis of proteins and nucleic acid fragments. Protein or nucleic acid bands containing as little as 1 or 2 ju,g can be detected by staining the gels after electrophoresis. 

Purified preparations of alkaline phosphatase from E. coli, judged homogeneous when examined in the analytical ultracentrifuge, contain several isozymes, because several bands which contain enzymic activity are obtained in starch-gel and disc-gel electrophoresis. Although most workers find three bands (38, 39, 41, 43, 69), four (44) and five (70) equally spaced bands have been found. 

Lazdunski and Lazdunski (43), separated three isozymes on DEAE-cellulose. They found that pure samples of either isozyme I (the isozyme with the least negative charge at pH 7.0 is referred to as isozyme I) or isozyme III, after dissociation and reassociation, gave only the original single band on disc-gel electrophoresis. Pure isozyme II, after dissociation and reassociation, gave three bands, corresponding to isozymes I, II, and III. It was later shown (44), that when monomers of isozymes I and III are mixed before reassociation, three bands are obtained. This... 

Figure 4. Poly acrylamide-disc gel electrophoretic patterns obtained for forms of CBH I from Trichoderma (2). The protein samples applied to these gels were, from left to right 20 fxg CBH I (A), 15 fig CBH I (B), 33 jig CBH I (C), and 40 fig CBH 1 (D). Forms A, B, and C were purified from a commercial T. viride cellulose preparation and Form D was purified from a culture of T. reesei QM 9123 grown on purified cellulose.

Figure 11. Polyacrylamide disc gel electrophoretic patterns of extracellular proteins produced by T. reesei QM 9414. The sample applied to the gel on the left was 130 fig extracellular protein from T. reesei my-celia grown on 1% Avicel (29), that applied to the gel on the right was 120 fig extracellular protein produced from sophorose-incubated mycelia. The bands shown here were stained for protein with Coomassie Blue and could, in all cases, also be stained for carbohydrate with the periodic acid-Schiff reagent.

The cellulase components that are synthesized in the presence of sophorose were investigated by the basic procedures previously described (1,2,4) for the isolation of cellulolytic components from commercial cellulase preparations. The purification to homogeneity of the proteins that yield the three predominant bands when the crude preparation is subjected to disc gel electrophoresis was accomplished by ion exchange chromatography. 

Figure 12. Polyacrylamide disc gel electrophoretic patterns of enzymes purified from the extracellular protein produced by T. reesei QM 9414 in response to ImM sophorose. To the gels, from left to right, were applied 175 fig extracellular protein mixture, 45 fig CBH II, 45 fig endoglucanase and 80 fig CBH I (D).

Figure 7. PAGE of reaction mixture after plasmin hydrolysis of B-casein for 0, 20, 30, 40, and 70 min (Slots 1-5) and Fractions 1 ana 2 from hydroxyapatite chromatography of hydrolysis products after plasmin treatment of fi-casein for 70 min (Slots 6 and 7). Fraction 2 contains a further phosphopeptide that is not visible in Slot 7 but appears on disc gels in the expected position for proteose peptone component 8F (cf. Ref. 32) (28).

Figure 9. Polyacrylamide disc gel electrophoresis of soluble SOD from (A) Spinacia (spinach) (a, b), Spirulina (blue-green alga) (c,d), ana from (B) rod outer segments from retina of cattle (a,b,d) and frogs (c,e) (39, 64)...

Myosin. Rabbit muscle myosin is a long, thin molecule (VI400 X 20-50 A) with a molecular weight of 5 X 10. It is composed of two heavy chains and four light chains as demonstrated by SDS-polyacrylamide disc gel electrophoresis. On tryptic digestion, myosin is split into the subunits, H-meromyosin (HMM) and L-mero-myosin (LMM). HMM is further split into S-l and S-2 subunits. While LMM is a rod of V)0% a-helical content, the a-helical content for HMM, S-l and S-2 fragments is 46%, 33% and 87%, respectively. The ATPase activity is localized in the S-l subunit (33,34). Although fish myosins appear to have the same structural profile (10,22,35-40) and similar amino acid composition as rabbit myosin (39,41,42), fish myosin is different from rabbit myosin in physicochemical properties such as solubility, viscosity and stability (10,22,35-40). 

However, our work on in vitro frozen storage of isolated carp actomyosin showed that actin is denatured progressively with myosin as demonstrated by SDS-polyacrylamide disc gel electrophoresis (90). 

Figure 9. Disc-gel electrophoretic patterns of 11S soybean globulin stored in a frozen or concentrated state, (a), original solution (b), after 2 days storage in a frozen state at —5°C (c), after the addition of 0.01 M mercaptoethanol (ME) to solution (b) (d), after 2 days of storage in a concentrated state (unfrozen) and (e), after the addition of 0.01M mercaptoethanol to solution (d) (lO).

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