Team:Freiburg/Protocols/SDS-PAGE
SDS-PAGE
SDS-PAGE, protein purification
remarks concerning the protocol (author, adapted from etc.)
material: Tris, glycin, SDS, glycerin, β-mercaptoethanol, bromphenol blue, methanol, acetic acid, coomassie brilliant blue, isopropyl alcohol …
duration: … min
| Buffers and solutions | |
|---|---|
| 10x SDS running buffer | 60 g Trisma (248 mM) |
| 288 g Glycin (2 M) | |
| 20 g SDS (1% (w/v)) | |
| ad 2 l H2O | |
| no need to pH, filter or degas | |
| dilute to 1X for running SDS-PAGE gels | |
| 4x SDS sample buffer | 40% Glycerin (w/v) |
| 20% β-mercaptoethanol (v/v) | |
| 9% SDS (v/v) | |
| 250 mM Tris (pH 6,8) | |
| 0.5% bromphenol blue (w/v) | |
| Coomassie staining solution | 225 ml methanol |
| 225 ml H20 | |
| 1.25 g Coomassie Brilliant Blue (CBB) R-250 (w/v) | |
| 50 ml (glacial) acetic acid | |
| destaining solution | 300 ml methanol (30% (v/v)) |
| 100 ml acetic acid (10% (v/v)) | |
| 600 ml H20 | |
| APS | 5 g APS |
| ad 50 ml H20 (10 % w/v final concentration | |
| make 1 ml aliquots, store at -20°C | |
| 4x sample loading buffer | 1.5 ml of 1 M Tris-HCl pH 6.8 |
| 3 ml of 1 M DTT (dithiothreitol) | |
| 0.6 g SDS | |
| 0.03 g bromphenol blue | |
| 2.4 ml glycerol | |
| ad 7.5 H2O | |
| if solution is orange/yellow in color, add 1 drops of 5 M NaOH to adjust pH | |
| make 500 µl aliquots and store at -20°C | |
| gel making buffer for resolving gel | 1.5 M Tris-HCl; pH 8.8 |
| 118 g Tris-HCl in H20 | |
| ad 500 ml H2O | |
| filter and degas | |
| gel making buffer for stacking gel | 1 M Tris-HCl; pH 6.8 |
| 78.8 g Tris-HCl in H20 | |
| ad 500 ml H2O | |
| filter and degas | |
Casting of gels:
| Resolving gels | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| amount of acrylamide | 8.50% | 10.50% | 12.50% | 15% | ||||||||||||
| number of gels | 1x | 2x | 3x | 4x | 1x | 2x | 3x | 4x | 1x | 2x | 3x | 4x | 1x | 2x | 3x | 4x |
| acrylamide (30%) [ml] | 1.4 | 2.8 | 4.3 | 5.7 | 1.7 | 3.3 | 5.0 | 6.7 | 2.1 | 4.2 | 6.3 | 8.4 | 2.5 | 5.0 | 7.5 | 10.0 |
| 1.5 mM Tris pH 8.8 [ml] | 1.3 | 2.5 | 3.8 | 5.0 | 1.3 | 2.5 | 3.8 | 5.0 | 1.3 | 2.5 | 3.8 | 5.0 | 1.3 | 2.5 | 3.8 | 5.0 |
| H20 [ml] | 2.3 | 4.7 | 7.0 | 9.3 | 2.1 | 4.2 | 6.2 | 8.3 | 1.7 | 3.3 | 5.0 | 6.6 | 1.3 | 2.5 | 3.8 | 5.0 |
| APS [µl] | 20 | 40 | 60 | 80 | 20 | 40 | 60 | 80 | 20 | 40 | 60 | 80 | 20 | 40 | 60 | 80 |
| TEMED [µl] | 6 | 12 | 18 | 24 | 6 | 12 | 18 | 24 | 6 | 12 | 18 | 24 | 6 | 12 | 18 | 24 |
| Stacking gel | ||||
|---|---|---|---|---|
| number of gels | 1x | 2x | 3x | 4x |
| acrylamide (30%) [µl] | 267 | 534 | 801 | 1068 |
| 0.5 mM Tris pH 6.8 [µl] 417 | 834 | 1251 | 1668 | |
| H20 [µl] | 967 | 1934 | 2901 | 3868 |
| APS [µl] | 25 | 50 | 75 | 100 |
| TEMED [µl] | 2.5 | 5 | 7.5 | 10 |
—- sds_page.pdf
