Team:TU Darmstadt/protocols/SDS-Page

From 2013.igem.org

(Difference between revisions)
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<ol>
<ol>
<br>
<br>
-
<B><li class=list1>Load and Run</li></B>
+
<B><li class=list1>Load & Run</li></B>
<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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<li>start the PAGE by applying 20 mA / gel at stacking</li>
<li>start the PAGE by applying 20 mA / gel at stacking</li>
<li>apply 40 mA / gel at separation </li>
<li>apply 40 mA / gel at separation </li>
 +
</ol>
<br>
<br>
-
<B><li class=list1>Staining of gel</li></B>
+
<ol>
-
<li>prepare the separating gel and fill it into the chamber </li>
+
<B><li class=list1>Staining & washing of gel</li></B>
-
<li>pour 1 mL isopropyle alcohol on the top of the gel to destroy air bubbles and prevent dehydration </li>
+
<li>disconnect glas plates containing the already run gel</li>
-
<li>discard the isoprpyl alcohol and pour the prepared stacking gel</li>
+
<li>cut off the stacking gel</li>
-
<li>stick in the comb</li>
+
<li>put the separation gel into the staining buffer and let it shake at room temperature for at least one hour</li>
-
<li>if not used, store the gel in wet cloth (to prevent dehydration) at 4°C</li>
+
<li>put the stained separation gel into the destaining buffer and let it shake for 5 minutes</li>
-
<li>if used, remove comb when the gel is solid and place it into SDS PAGE chamber</li>
+
<li>repeat the previous step at least twice again with fresh destaining buffer each</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>
</ol>
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Revision as of 19:36, 4 October 2013







Labbook | Matierals | Protocols

Protocols


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

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

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