Equipment:

- Two glass plates

- Gel caster

- Comb

- VWR Power Source 300V

- Heat block

Chemicals & consumables:

- SDS

- Rotiphorese® (30%)

- Tris HCl

- Glycine

- TEMED

- APS

- ddH₂O

- Isopropyl alcohol

- Glycerine

- Beta-Mercaptoethanol

- Bromphenolblue

- Coomassie brilliant blue G250

- Coomassie brilliant blue R250

- Methanol

- Acetate (99%)

Buffers & gels:

- Separation buffer (0.5 M Tris, 0.4% SDS, pH = 8.8)

- Stacking buffer (0.5 M Tris, 0.4% SDS, pH = 6.6)

- Running buffer (0.25 M Tris, 2 M glycine, 1% SDS , pH = 8.3)

- Separation gel 12.5% (5 ml Separation buffer, 6.25 ml Rotiphorese®, 3.75 ml Aqua dest., 30 µl TEMED, 30 µl APS (40%))

- Stacking gel 4% (3 ml Separation buffer, 1.33 ml Rotiphorese®, 5.67 ml Aqua dest.,20 µl TEMED, 20 µl APS (40%)

- 3x Sample buffer (65 mM Tris, 4% SDS, 20% glycerine, 10% beta-Mercaptoethanol, 1 tip of bromphenolblue, pH = 6.75)

- Staining buffer (0.5 g Coomassie brilliant blue G250 & R250 each, 100 ml methanol, 100 ml Aqua dest., 20 ml acetate)

- Destaining buffer (400 ml Aqua dest., 100 ml methanol, 30 ml acetate)

Procedure:

Load & Run

- prepare the separating gel and fill it into the chamber

- pour 1 mL isopropyle alcohol on the top of the gel to destroy air bubbles and prevent dehydration

- discard the isoprpyl alcohol and pour the prepared stacking gel

- stick in the comb

- if not used, store the gel in wet cloth (to prevent dehydration) at 4°C

- if used, remove comb when the gel is solid and place it into SDS PAGE chamber

- fill chamber with running buffer

- after heating the samples with 3x sample buffer at 70°C for 15 min, apply 20 µl to each pocket

- load outer pocket with a commercial protein marker

- start the PAGE by applying 20 mA / gel at stacking

- apply 40 mA / gel at separation

Staining & washing of gel

- disconnect glas plates containing the already run gel

- cut off the stacking gel

- put the separation gel into the staining buffer and let it shake at room temperature for at least one hour

- put the stained separation gel into the destaining buffer and let it shake for 5 minutes

- repeat the previous step at least twice again with fresh destaining buffer each