Separation of Native Proteins in an Ultra Thin Support Bound Porosity Gradient

To prepare a thin gradient gel of the dimensions 120 x 250 x 0.5 mm fixed to a derivatized clear and flexible polyester foil (e.g. manufactured by Gel

Bond, Marine Colloids, Rockland, MN, USA or Serva, Heidelberg, Germany), the gel-forming devices shown in Figure 6 may be used. (When the cassettes are assembled the slot formers must not touch the opposite glass wall but leave a space of 0.1 mm in between). The following solutions may be used to form a gradient ranging from 3 to 30% T:

1. gel buffer: 90 mmol L — Tris, 80 mmol L — boric acid, 2.5 mmol L-1 EDTA-Na2, pH 8.4;

2. electrode buffer: 1 in 2 diluted gel buffer;

3. stock acrylamide solution (30% T: 28.8 g acryl-amide plus 1.2 g Bis plus 50 mL gel buffer, made to 100 mL with distilled water);

Figure 4 Vertical electrophoretic apparatus in which up to four glass cassette-cast porosity gradient gels can be inserted. Upper electrode vessel with 2 rubber gaskets to hold 2 to 4 glass cassettes, each containing a porosity gradient made of PA; # , - , electrodes. Modified from an instruction leaflet published by Pharmacia, Uppsala, Sweden.

Figure 4 Vertical electrophoretic apparatus in which up to four glass cassette-cast porosity gradient gels can be inserted. Upper electrode vessel with 2 rubber gaskets to hold 2 to 4 glass cassettes, each containing a porosity gradient made of PA; # , - , electrodes. Modified from an instruction leaflet published by Pharmacia, Uppsala, Sweden.

Figure 5 Electrophoresis of plant diaphorase isoenzymes in a 4-20% T PA gradient gel of 0.8 mm thickness (length 175 mm, height 75 mm). (A) Zymogram of diaphorase enzymes (numbers indicate genotypes of the tetrameric enzyme at locus B. (B) Schematic representation of genotypes at locus DIA-A and DIA-B. Enzyme source: leaf buds of seven different trees of European beech (Fagus sylvatica L.). Conditions of electrophoresis: gel and electrode buffer: 45 mmol L~1 Tris, 40mmolL~1 boric acid, 1.25 mmol L~1 EDTA-Na2; pH 8.4; running time 4h; voltage gradient 40 Vcm~1; temperature 5°C.

Enzyme extraction: 1.5 mL Eppendorf tubes containing 150 mg of green bud leaves, 50 mg of quartz sand and 600 ^L of extraction medium were cooled from underneath with ice water. A motor-driven grinding cone adapted in the shape of the tube (rotating at 700 rpm) was used to homogenize the material. The extraction medium contained in 100 mL: 1.21 g Tris, 1.43 g Na2HPO4, 60 mg l-cysteine, 210 mg ascorbic acid, 14 g sucrose, 40 mg NADP, 15 g polyclar AT (PVPP) and 1 g polyethylene glycol, pH 7.5 (with H3PO4). The homogenate was centrifuged for 30 min at 4°C and 10000 g and the clear supernatant used as crude enzyme extract. Samples of 8 ^L were applied per lane. Diaphorase isozymes were visualized histochemically (60 mL 25 mmol L~1 Tris-HCl, pH 8.5, containing 24mg NADH, 1.5 mg 2,5 dichlorphenolindophenol-Na x2H2O (DCPIP) and 1.8 mL MTT (500 mg 100mL~1 aq. bidest. water). Anode at bottom. A, Enzymes of gene locus DIA-A; B, locus DIA-B. In (A) not all genotypes indicated in (B) are shown.

Figure 5 Electrophoresis of plant diaphorase isoenzymes in a 4-20% T PA gradient gel of 0.8 mm thickness (length 175 mm, height 75 mm). (A) Zymogram of diaphorase enzymes (numbers indicate genotypes of the tetrameric enzyme at locus B. (B) Schematic representation of genotypes at locus DIA-A and DIA-B. Enzyme source: leaf buds of seven different trees of European beech (Fagus sylvatica L.). Conditions of electrophoresis: gel and electrode buffer: 45 mmol L~1 Tris, 40mmolL~1 boric acid, 1.25 mmol L~1 EDTA-Na2; pH 8.4; running time 4h; voltage gradient 40 Vcm~1; temperature 5°C.

Enzyme extraction: 1.5 mL Eppendorf tubes containing 150 mg of green bud leaves, 50 mg of quartz sand and 600 ^L of extraction medium were cooled from underneath with ice water. A motor-driven grinding cone adapted in the shape of the tube (rotating at 700 rpm) was used to homogenize the material. The extraction medium contained in 100 mL: 1.21 g Tris, 1.43 g Na2HPO4, 60 mg l-cysteine, 210 mg ascorbic acid, 14 g sucrose, 40 mg NADP, 15 g polyclar AT (PVPP) and 1 g polyethylene glycol, pH 7.5 (with H3PO4). The homogenate was centrifuged for 30 min at 4°C and 10000 g and the clear supernatant used as crude enzyme extract. Samples of 8 ^L were applied per lane. Diaphorase isozymes were visualized histochemically (60 mL 25 mmol L~1 Tris-HCl, pH 8.5, containing 24mg NADH, 1.5 mg 2,5 dichlorphenolindophenol-Na x2H2O (DCPIP) and 1.8 mL MTT (500 mg 100mL~1 aq. bidest. water). Anode at bottom. A, Enzymes of gene locus DIA-A; B, locus DIA-B. In (A) not all genotypes indicated in (B) are shown.

4. dense acrylamide solution (30% T: to 6.5 mL stock solution is added, shortly before use, 20 |L TEMED (1 in 10 with H2O diluted solution) and 5 |L ammonium persulfate solution (40% w/v in distilled water));

5. light acrylamide solution (3% T: 1 vol of stock acrylamide solution is diluted with 4.5 vol distilled water and 4.5 vol of gel buffer shortly before use and 40 |L TEMED (1 in 10 with distilled water diluted solution) and 10 | L ammonium persulfate

Figure 6 Preparation of an ultra-thin PA gradient gel fixed to a polyester foil. (A) Rolling the polyester foil (reactive side up, e.g. Gel Bond) on to one of the glass plates used to build the casting glass cassette: 1, levelling table; 2, glass plate; 3, hydrophilic side of polyester foil; 4, water layer; 5, rubber roller. (B) Trough template preparation. The bars are prepared from two layers of self-adhesive tape with a scalpel. (C) Assembling the glass cassette to cast the PA gradient. (D) Casting the porosity gradient: two-chamber mixer and glass cassette. Reproduced with permission from Rothe (1991).

Figure 6 Preparation of an ultra-thin PA gradient gel fixed to a polyester foil. (A) Rolling the polyester foil (reactive side up, e.g. Gel Bond) on to one of the glass plates used to build the casting glass cassette: 1, levelling table; 2, glass plate; 3, hydrophilic side of polyester foil; 4, water layer; 5, rubber roller. (B) Trough template preparation. The bars are prepared from two layers of self-adhesive tape with a scalpel. (C) Assembling the glass cassette to cast the PA gradient. (D) Casting the porosity gradient: two-chamber mixer and glass cassette. Reproduced with permission from Rothe (1991).

solution (40% w/v in distilled water) is added. The gradient is made of 6.5 mL of dense acrylamide solution and 6.5 mL of light acrylamide solution. After gradient formation, 2 mL of light acrylamide solution is overlaid; the slots must be situated in the middle of the 3% T range.

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