From 2013.igem.org
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| | <h2><font size="6" color="#F0F8FF" face="Arial regular">SDS PAGE</font></h2> | | <h2><font size="6" color="#F0F8FF" face="Arial regular">SDS PAGE</font></h2> |
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| - | <p text-aligne:left style="margin-left:50px; margin-right:50px"> | + | <p text-aligne:left style="margin-left:50px; margin-right:50px"><font size="4.5" color="#F0F8FF" face="Arial regular"> |
| | + | <B> Materials<br></B></font></p> |
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| - | <B><li class=list1>Load & Run</li></B> | + | <B><li class=list1>Load & Run</li></B></ol><ol> |
| | <li>prepare the separating gel and fill it into the chamber </li> | | <li>prepare the separating gel and fill it into the chamber </li> |
| | <li>pour 1 mL isopropyle alcohol on the top of the gel to destroy air bubbles and prevent dehydration </li> | | <li>pour 1 mL isopropyle alcohol on the top of the gel to destroy air bubbles and prevent dehydration </li> |
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| | <br> | | <br> |
| | <ol> | | <ol> |
| - | <B><li class=list1>Staining & washing of gel</li></B> | + | <B><li class=list1>Staining & washing of gel</li></B></ol><ol> |
| | <li>disconnect glas plates containing the already run gel</li> | | <li>disconnect glas plates containing the already run gel</li> |
| | <li>cut off the stacking gel</li> | | <li>cut off the stacking gel</li> |
Revision as of 21:57, 4 October 2013
SDS PAGE
Materials
Equipment
- - Two glass plates
- - Gel caster
- - Comb
- - VWR Power Source 300V
- - Heat block
Chemicals & consumables
- - SDS
- - Rotiphorese® (30%)
- - Tris HCl
- - Glycine
- - TEMED
- - APS
- - Aqua dest.
- - Isopropyle 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 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
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