Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

Transcription

1 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) mini-gel type <METHODS> A. Preparation of separating gel 1. Mix distilled water, SDS, acrylamide-bis stock and 1.5 M Tris-HCl to give the correct % according to the attached list. 2. Degas for 15 minutes. In the meantime, thaw out an aliquot of APS or prepare 10% APS solution freshly. 3. Assemble gel sandwich (plates, spacers, clamps). Mark level to which separating gel will come (about 0.5 cm below the teeth of the comb). 4. Prepare syringe with attached needle and fill with distilled water or water saturated butanol. 5. After degassing, disconnect the vacuum pump and transfer gel mixture to a beaker. Add correct amount of APS and TEMED and mix by swirling gently. 6. Pour gel directly from the beaker (or by using a pipette) to the marked level. Very carefully, apply overlay (water or water saturated butanol) by the syringe to give a layer 5 mm thick. 7. After polymerisation (ideally minute-slow or fast polymerisation can be modified by adjusting APS and TEMED), remove butanol, rinse and apply a layer of water when butanol is used as overlay. If saparating gels are being left overnight, water should be added up to the top of the plates. The plates should be covered with cling film and kept at 4 C. 8. If gel is to be run immediately, the overlay should be removed and stacking gel poured. 74

2 B. Preparation of stacking gel 1. Mix distilled water, acrylamide-bis, SDS and 0.5 M Tris-HCl according to the attached sheet. 2. Degas for 15 minutes 3. Remove from vacuum and transfer to a beaker. Add correct amount of APS and TEMED, swirl gently to mix. 4. After removing overlay, pour stacking gel until a level is 2-3 mm below top of plates. 5. Gently insert comb into top of sandwich at an angle to prevent air bubbles being trapped below the comb teeth. Leave to polymerise. C. Preparation of samples 1. Each lane should contain 5-10 µg protein depending on the complexity of the sample. 2. Dilute or concentrate sample to give correct amount of protein in 5 to 10 µl and add 1 part sample buffer to 1 part sample. Sample buffer with DTT is for reducing form and without DTT is for non-reducing form. Aluminium foil 3. Heat in steam of boiling water for 5 minutes in Eppendorf tubes (closed but with a pin-hole in the top). Cool and load gel. # The final sample (mixture of sample and sample buffer) must contain 5-10 µg protain in 5-10 µl. For instance, if the sample is crude, prepare 10 µl of 4 mg/ml sample and add 10 µl of sample buffer. Boil the mixture and apply 5 µl/well to gel, which contains 10 µg protein. Or add 10 µl of sample buffer to 10 µl of 2 mg/ml sample and load 10µl of the mixture/well. 75

3 D. Loading gel 1. Remove comb from stacking gel by carefully lifting straight upwards when polymerised. 2. Fill wells with running buffer and load gel (5-10 µl/well), starting with 5 µl of sample buffer (S.B.) in left and right end wells. Then, load markers (M.W.) and samples as quickly as posiible to avoid diffusion. M.W. Sample M.W. 3. Fill upper and lower buffer chambers with running buffer, connect power supply and commence running the gel. E. Running condition 50 V (constant voltage) until tracking dye has passed through the stacking gel (for about 20 minutes), then 100 V. F. After running gel 1. When tracking dye is about to run off the bottom of the gel, turn off power pack, disconnect leads and remove gel sandwich. 2. Carefully separate plates by gently levering apart using the spacers and cut off the stacking gel. 3. If the gel is to be stained, it should be placed in fix/destain solution (silver stain) or directly into Coomassie Blue staining solution. If the gel is to be blotted, the gel should be placed in transfer buffer (see blotting sheet). 76

4 G. Coomassie Blue staining 1. After electrophoresis, the gel is placed in a dish containing Coomassie Blue stain. 2. After minutes, the stain is poured off for re-use and the gel rinsed in destain. The gel should then be soaked in several changes of a large volume of destain on a rocking platform until the background of the gel is clear. 3. The gel can then be photographed and/or dried. Before drying, the gel should be soaked for one hour in drying reagent. H. Gel ingredients 1. Separating gel % Acrylamide 8% 10% 11% 12% 15% 1.5 M Tris-HCl % SDS Acrylamide stock Distilled water APS TEMED Stacking gel % acrylamide 3% 5% 0.5M Tris-HCl % SDS Acrylamide stock Distilled water APS TEMED

5 <SOLUTIONS> # 30% acrylamide/bis stock Acrylamide g N-N bis methylene acrylamide g Dissolve in 50 ml distilled water, QS to 100 ml and store at 4 C for a month (use Sigma product No. A6050). # 1.5 M Tris-HCl, ph 8.8 (for separating gel) Dissolve g Tris in 60 ml of distilled water, adjust ph to 8.8 with concentrated HCl, then QS to 100 ml. Store at 4 C. # 0.5 M Tris-HCl, ph 6.8 (for stacking gel) Dissolve 6 g Tris in 60 ml of distilled water, adjust ph to 6.8 with concentrated HCl, then QS to 100 ml. Store at 4 C. # 10% SDS Dissolve 10 g SDS to distilled water and QS to 100 ml. Store at room temperature. # Running buffer, ph 8.3 (10X concentrate) Tris g Glycine g SDS g Dissolve in 700 ml of distilled water and QS to 1 l (add 1 part to 9 parts distilled water for working strength). Store at room temperature. # Sample cocktail (20 ml) 0.5M Tris-HCl, ph ml 10% SDS ml Glycerol ml Distilled water ml Store at room temperature. # Bromophenol Blue/DTT Dithiothreitol g Bromophenol Blue... 6 mg Dissolve in 6 ml of distilled water. Store frozen in 0.1 ml aliquots. 78

6 # Sample buffer * For reducing condition Add 0.65 ml sample cocktail to 0.1 ml of Bromophenol Blue/DTT just before use. * For non-reducing condition Add 0.1 ml of 1 mg/ml Bromophenol Blue solution to 0.65 ml sample cocktail. Stable at room temperature. # Fix/destain solution (1 l) Methanol ml Glacial acetic acid ml Distilled water ml Store at room temperature. # Coomassie Blue stain Dissolve 1 g Coomassie Blue R 250 in 1 l of fix/destain solution and filter (solution can be re-used). Store at room temperature. # Drying reagent (1 l) Glacial acetic acid ml glycerol ml Distilled water ml Store at room temperature. # 10% ammonium persulphate (APS) Dissolve 2 g APS in 20 ml of distilled water and aliquot in 450 µl lots. Store at -20 C. 79