Team:Wageningen UR/Protocol/SDSPolyacrylamide

From 2012.igem.org

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= Purification of the VLPs =
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= SDS-polyacrylamide and native gel electrophoresis =
== Reagents & Materials ==
== Reagents & Materials ==
== Procedure ==
== Procedure ==
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*Wear gloves
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*Clean glass slides with MQ and dry
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'''Running gel'''
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<ol>
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<li>Prepare running gel mix in a small beaker for 2 gels:</li>
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*4 ml MQ
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*3.3 ml 30% acrylamide mix
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*2.5 ml 1.5 M TRIS pH 8.8
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*0.1 ml 10% SDS
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*0.1 ml 10% ammonium persulphate
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<li>Places glass slides in holders and tighten in rack.</li>
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polymerization:
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<li>Add 4 ul TEMED to the running gel mix, mix and pipet the gel into the slides until the green edge.</li>
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<li>Add a layer of iso-propanol on top of the polymerizing gel to equalize.</li>
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</ol>
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'''Stacking gel'''
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 +
<ol>
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<li>When the running gel has fully polymerized, remove the remaing iso-propanol with a tissue</li>
 +
<li>Prepare stacking gel mix in a small beaker for 2 gels:</li>
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*1.4 ml MQ
 +
*0.33 ml 30% acrylamide mix
 +
*0.25 ml 1 M TRIS pH 6.8
 +
*0.02 ml 10% SDS
 +
*0.02 ml 10% ammonium persulphate
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<li>Add 2 ul TEMED to the stacking gel mix, mix and pipet the gel onto the running gel.</li>
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<li>Place comb</li>
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<li>After the stacking gel has fully polymerized, the gels can be stored at 4 degree Celcius in a box with wet tissues.</li>
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</ol>
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'''Sample preparation'''
 +
 +
Protein denaturation for SDS-PAGE:
 +
<ol>
 +
<li>Dependent on the protein concentration, typically 15 ul of sample are transferred to a 1.5 ml tube</li>
 +
<li>Pipet 5 ul of SDS-PAGE loading buffer 5X in the caps of the tubes</li>
 +
<li>Spin tubes briefly</li>
 +
<li>Heat samples and a protein marker in a thermobloch 10 minutes at 99  degree Celcius</li>
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<li>Spin down samples briefly</li>
 +
</ol>
 +
 +
 +
'''Running samples'''
 +
 +
<ol>
 +
<li>Place the gels in 1X SDS running buffer and remove combs</li>
 +
<li>Pipet a maximum of 20uL of sample in the slots of the gel using long tips</li>
 +
<li>Connect to power supply and run at 19 mA per gel</li>
 +
<li>When the dye has reached the running gel, power can be increased</li>
 +
<li>Run the samples until the lower green edge</li>
 +
</ol>
 +
 +
 +
'''Staining'''
 +
 +
<ol>
 +
<li>Rinse gels with MQ and lift the top glass with wedge and cut the edges</li>
 +
<li>Carefully place gels in Coomassie staining solution for 15 minutes</li>
 +
<li>Recover the staining solution with a funnel</li>
 +
<li>Rinse gels with MQ</li>
 +
<li>Place gels in de-staining solution with tissues on each side for 15 minutes</li>
 +
<li>De-stain gel until bands are clearly visible</li>
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</ol>

Latest revision as of 13:38, 24 September 2012


SDS-polyacrylamide and native gel electrophoresis

Reagents & Materials

Procedure

  • Wear gloves
  • Clean glass slides with MQ and dry


Running gel

  1. Prepare running gel mix in a small beaker for 2 gels:
    • 4 ml MQ
    • 3.3 ml 30% acrylamide mix
    • 2.5 ml 1.5 M TRIS pH 8.8
    • 0.1 ml 10% SDS
    • 0.1 ml 10% ammonium persulphate
  2. Places glass slides in holders and tighten in rack.

    3. polymerization:

  3. Add 4 ul TEMED to the running gel mix, mix and pipet the gel into the slides until the green edge.
  4. Add a layer of iso-propanol on top of the polymerizing gel to equalize.


Stacking gel

  1. When the running gel has fully polymerized, remove the remaing iso-propanol with a tissue
  2. Prepare stacking gel mix in a small beaker for 2 gels:
    • 1.4 ml MQ
    • 0.33 ml 30% acrylamide mix
    • 0.25 ml 1 M TRIS pH 6.8
    • 0.02 ml 10% SDS
    • 0.02 ml 10% ammonium persulphate
  3. Add 2 ul TEMED to the stacking gel mix, mix and pipet the gel onto the running gel.
  4. Place comb
  5. After the stacking gel has fully polymerized, the gels can be stored at 4 degree Celcius in a box with wet tissues.


Sample preparation

Protein denaturation for SDS-PAGE:

  1. Dependent on the protein concentration, typically 15 ul of sample are transferred to a 1.5 ml tube
  2. Pipet 5 ul of SDS-PAGE loading buffer 5X in the caps of the tubes
  3. Spin tubes briefly
  4. Heat samples and a protein marker in a thermobloch 10 minutes at 99 degree Celcius
  5. Spin down samples briefly


Running samples

  1. Place the gels in 1X SDS running buffer and remove combs
  2. Pipet a maximum of 20uL of sample in the slots of the gel using long tips
  3. Connect to power supply and run at 19 mA per gel
  4. When the dye has reached the running gel, power can be increased
  5. Run the samples until the lower green edge


Staining

  1. Rinse gels with MQ and lift the top glass with wedge and cut the edges
  2. Carefully place gels in Coomassie staining solution for 15 minutes
  3. Recover the staining solution with a funnel
  4. Rinse gels with MQ
  5. Place gels in de-staining solution with tissues on each side for 15 minutes
  6. De-stain gel until bands are clearly visible
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