Team:TU Darmstadt/protocols/Protein Expression

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Protein Expression

Equipment & chemicals

Chemicals & consumables

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 most 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