Native Gel Assay for ADH Activity

Changes made according to hand-written annotations on previous copies indicated by italics

Jim Sloan

September 1994

 

ADH lysis buffer + glycerol

            85 mM KCl

                  30 mM TRIS pH 7.5

                  3 mM Mg acetate

                  25% glycerol

 

5x Loading Buffer                                                                Running Buffer

                  50 mM TRIS pH 6.8                                                                                   1.44% glycine

                  25% Glycerol                                                                                                 0.3% TRIS base

                  0.05% bromophenol blue              

 

Native Separating Gel (10 ml)

            5.7 ml H2O

                  2.5 ml of 1.5 M TRIS pH 8.8

                  1.7 ml of 30% acrylamide/bisacrylamide (37.5:1)

                  50 ml of 10% ammonium persulfate

                  5 ml TEMED, overlay w/ H2O

 

Native Stacking Gel (5 ml)

                  3.17 ml H2O

                  1.25 ml of 0.5 M TRIS pH 6.8

                  500 ml of 30% acrylamide/bisacrylamide (37.5:1)

                  25 ml of 10% ammonium persulfate

                  5 ml TEMED

 

100 mM TRIS pH 8.8

 

ADH Activity Buffer (50 ml)

                  46.7 ml H2O

                  3.3 ml of 1.5 M TRIS pH 8.8

                  50 mg NAD

                  10 mg nitroblue tetrazolium

                  4 mg phenazine methosulfate

                  50 ml of 95% ethanol

 

Yeast Culture

1. Grow a yeast culture in 5-10 ml to an absorbance of about 0.7-1.5 OD600

 

2.  Pellet the cells and discard the supernatant

 

3.  Wash the cells with 0.7 ml of ADH lysis buffer + glycerol.  Pellet the cells, discard the supernatant and freeze the cells on dry ice, then at -70oC

 

(To keep ADH2 repressed, make sure they have gone through many doublings in repressing conditions.  We use 5% glucose medium and at least 6-8 doublings (10 if you are being conservative).  For strains like multi-copy ADR1 that derepress easily, keep the OD600 under 0.7.  To derepress, wash the cells and transfer to low glucose, e.g. 0.05% glucose medium).

 

Protein Extraction and Electrophoresis

Changes made according to hand-written annotations on earlier copies indicated by italics.

 

1. Prepare the native gel using the minigel apparatus. Use thick (1.5mm) spacers; combs = 10 wells; 10 ml = 1 gel. When the gel has polymerized, set the gel in the cold room or chill on ice. Put 1x Tis-glycine at 4¡C. When you pour the gel, do not overlay w/ butanol -- use H2O w/ pasteur pipet.

 

2. Thaw the cells on ice. Add 0.2 gm glass beads and 200 ml ADH buffer + glycerol containing 0.07% 2-mercaptoethanol.

                  * OR add glass beads equivalent to pellet volume, add LYSIS buffer to ~2-3mm above pellet and beads.

 

3. Grind the cells in the vortex mixer at 4¡C. Agitate in three intervals, 2 minutes on and 2 minutes off.

 

4. Spin down the beads and debris at 4¡C. Full speed, 10Õ, transfer to new tube.

 

5. Determine the protein concentration of the extract in the mg/ml (using Bio-Rad Protein Assay kit or the Lowry method). Also 10 mg/ml BSA.

 

6. Mix 50-100 mg or more of total protein with 1/5 volume of the loading buffer for each well and load. 15-25 mg if a lot of AdhII is expected (DR cells); 50-100 mg if little AdhII is expected.

 

7. Run the gel in the cold room or on ice at 150 V until the blue dye reaches the bottom of the gel

Tris-glycine 10x is in the cold room.

 

8. Remove the gel from the plates and soak it in 50 ml 100mM TRIS pH 8.8 at room temperature for 5 minutes.

 

9. Pour off the TRIS solution and add 50 ml ADH activity buffer. Incubate at 37¡C until the desired staining is obtained, about 10Õ.

 

10. Pour off the ADH activity buffer and rinse the gel with H2O. The gel can be left soaking in H2O.

 

11. The gel can be preserved by drying it between sheets of cellophane.

 

Keep everything cold!

 

Dilute Bio-Rad reagent 5x. Put 1 ml in each sample tube plus 10 for standard curve and blank(s). Add 1-10 ml cell extract (or try 2x). Standard curve is 1, 2, 3É. 10 ml 1mg/ml BSA (10 mg/ml from restriction enzymes). Vortex, read OD595. Do all samples in duplicate.