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Team:TU Darmstadt/Notebook/Methods/SDS-PAGE
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Revision as of 15:54, 16 October 2014
SDS-PAGE
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
