Team:TU Darmstadt/protocols/SDS-Page

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<br>
<br>
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<h2><font size="6" color="#F0F8FF" face="Arial regular">Protocols</font></h2>
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<h2><font size="6" color="#F0F8FF" face="Arial regular">SDS PAGE</font></h2>
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<font size="3" color="#F0F8FF" face="Arial regular">
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<p text-aligne:left style="margin-left:50px; margin-right:50px">
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<p text-aligne:left style="margin-left:50px; margin-right:50px"><font size="4.5" color="#F0F8FF" face="Arial regular">
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<B> Materials<br></B></font></p>
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 +
 
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<p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular">
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<br>
 +
<B>Equipment<br></B>
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<div align="left" style="margin-left:60px; margin-right:50px">
 +
<ul>
 +
<li class=list1>- Two glass plates</li>
 +
<li class=list1>- Gel caster</li>
 +
<li class=list1>- Comb</li>
 +
<li class=list1>- VWR Power Source 300V</li>
 +
<li class=list1>- Heat block</li>
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</ul>
 +
</div>
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</font>
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</p>
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 +
<p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular">
 +
<br>
 +
<B>Chemicals & consumables<br></B>
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<div align="left" style="margin-left:60px; margin-right:50px">
 +
<ul>
 +
<li class=list1>- SDS</li>
 +
<li class=list1>- Rotiphorese® (30%)</li>
 +
<li class=list1>- Tris HCl</li>
 +
<li class=list1>- Glycine</li>
 +
<li class=list1>- TEMED</li>
 +
<li class=list1>- APS</li>
 +
<li class=list1>- Aqua dest.</li>
 +
<li class=list1>- Isopropyle alcohol</li>
 +
<li class=list1>- Glycerine</li>
 +
<li class=list1>- Beta-Mercaptoethanol</li>
 +
<li class=list1>- Bromphenolblue</li>
 +
<li class=list1>- Coomassie brilliant blue G250</li>
 +
<li class=list1>- Coomassie brilliant blue R250</li>
 +
<li class=list1>- Methanol</li>
 +
<li class=list1>- Acetate (99%)</li>
 +
</ul>
 +
</div>
 +
</font>
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</p>
 +
 
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<p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular">
 +
<br>
 +
<B>Buffers & gels<br></B>
 +
<div align="left" style="margin-left:60px; margin-right:50px">
 +
<ul>
 +
<li class=list1>- Separation buffer (0.5 M Tris, 0.4% SDS, pH = 8.8)</li>
 +
<li class=list1>- Stacking buffer (0.5 M Tris, 0.4% SDS, pH = 6.6)</li>
 +
<li class=list1>- Running buffer (0.25 M Tris, 2 M glycine, 1% SDS , pH = 8.3)</li>
 +
<li class=list1>- Separation gel 12.5% (5 ml Separation buffer, 6.25 ml Rotiphorese®, 3.75 ml Aqua dest., 30 µl TEMED, 30 µl APS (40%))</li>
 +
<li class=list1>- Stacking gel 4% (3 ml Separation buffer, 1.33 ml Rotiphorese®, 5.67 ml Aqua dest.,20 µl TEMED, 20 µl APS (40%)</li>
 +
<li class=list1>- 3x Sample buffer (65 mM Tris, 4% SDS, 20% glycerine, 10% beta-Mercaptoethanol, 1 tip of bromphenolblue, pH  = 6.75)</li>
 +
<li class=list1>- Staining buffer (0.5 g Coomassie brilliant blue G250 & R250 each, 100 ml methanol, 100 ml Aqua dest., 20 ml acetate)</li>
 +
<li class=list1>- Destaining buffer (400 ml Aqua dest., 100 ml methanol, 30 ml acetate)</li>
 +
</ul>
 +
</div>
 +
</font>
 +
</p>
 +
<br>
 +
<p text-aligne:left style="margin-left:50px; margin-right:50px"><font size="4.5" color="#F0F8FF" face="Arial regular">
 +
<B> Procedure<br></B></font></p>
 +
<p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular">
 +
 
 +
 
 +
<div align="left" style="margin-left:30px; margin-right:50px">
 +
<ol>
 +
<br>
 +
<B><li class=list1>Load & Run</li></B></ol><ol>
 +
<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>discard the isoprpyl alcohol and pour the prepared stacking gel</li>
 +
<li>stick in the comb</li>
 +
<li>if not used, store the gel in wet cloth (to prevent dehydration) at 4°C</li>
 +
<li>if used, remove comb when the gel is solid and place it into SDS PAGE chamber</li>
 +
<li>fill chamber with running buffer</li>
 +
<li>after heating the samples with 3x sample buffer at 70°C for 15 min, apply 20 µl to each pocket</li>
 +
<li>load most outer pocket with a commercial protein marker</li>
 +
<li>start the PAGE by applying 20 mA / gel at stacking</li>
 +
<li>apply 40 mA / gel at separation </li>
 +
</ol>
 +
<br>
 +
<ol>
 +
<B><li class=list1>Staining & washing of gel</li></B></ol><ol>
 +
<li>disconnect glas plates containing the already run gel</li>
 +
<li>cut off the stacking gel</li>
 +
<li>put the separation gel into the staining buffer and let it shake at room temperature for at least one hour</li>
 +
<li>put the stained separation gel into the destaining buffer and let it shake for 5 minutes</li>
 +
<li>repeat the previous step at least twice again with fresh destaining buffer each</li>
 +
</ol>
 +
</div>
 +
</font></p>

Latest revision as of 00:38, 5 October 2013







Lab book | Materials | Protocols

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


  1. Load & Run
  1. prepare the separating gel and fill it into the chamber
  2. pour 1 mL isopropyle alcohol on the top of the gel to destroy air bubbles and prevent dehydration
  3. discard the isoprpyl alcohol and pour the prepared stacking gel
  4. stick in the comb
  5. if not used, store the gel in wet cloth (to prevent dehydration) at 4°C
  6. if used, remove comb when the gel is solid and place it into SDS PAGE chamber
  7. fill chamber with running buffer
  8. after heating the samples with 3x sample buffer at 70°C for 15 min, apply 20 µl to each pocket
  9. load most outer pocket with a commercial protein marker
  10. start the PAGE by applying 20 mA / gel at stacking
  11. apply 40 mA / gel at separation

  1. Staining & washing of gel
  1. disconnect glas plates containing the already run gel
  2. cut off the stacking gel
  3. put the separation gel into the staining buffer and let it shake at room temperature for at least one hour
  4. put the stained separation gel into the destaining buffer and let it shake for 5 minutes
  5. repeat the previous step at least twice again with fresh destaining buffer each

Retrieved from "http://2013.igem.org/Team:TU_Darmstadt/protocols/SDS-Page"