Team:TU Darmstadt/protocols/SDS-Page

From 2013.igem.org

(Difference between revisions)

Revision as of 19:27, 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

Load and 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 of gel

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

@@ Line 192: / Line 192: @@
 <li class=list1>- Comb</li>
 <li class=list1>- VWR Power Source 300V</li>
+<li class=list1>- Heat block</li>
 </ul>
 </div>
@@ Line 213: / Line 214: @@
 <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>
@@ Line 229: / Line 234: @@
 <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>
@@ Line 243: / Line 248: @@
 <div align="left" style="margin-left:30px; margin-right:50px">
 <ol>
+<br>
+<B><li class=list1>Load and Run</li></B>
 <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>
@@ Line 248: / Line 255: @@
 <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>
+<br>
+<B><li class=list1>Staining of gel</li></B>
+<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>
 </div>
 </font></p>