Team:Calgary/Notebook/Protocols/Immunoprecipitation

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<h1>Immunoprecipitation</h1>
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<p>Materials: </p>
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<ul> <li>Cell lysates</li>
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<li>Protein A conjugated agarose beads</li>
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<li>Antibody of interest:0.5-1ug/ reaction</li>
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<li>lysis buffer: 50 mM NaH2PO4
300 mM NaCl
10 mM imidazole
Adjust pH to 8.0 using NaOH. </li>
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<li>0.5 mm glass beads</li>
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<li>Rotator</li>
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<li>All reagents to run a western blot</li></ul>
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<ol> <li>Grow up overnight cultures in the correct selection marker. </li>
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<li>Spin down the pellets next morning and lyse the cells using bead beaters</li>
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<ul><li> Incubate sample with glass beads and lysis buffer containing 100uM protease inhibitor for 5 mins on ice and then shake for 5 minutes on a bead beater. Repeat twice.</ul><li>
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<li>Do a Bradford assay to determine the protein concentration of your samples<li>
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<li>Combine 500ug-1000ug of each lysate</li>
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<ul><li>Amount of protein you add for each lysate will vary upon the abundance of your protein of interest in the lysate</li></ul>
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<li>Add the antibody (0.5-1ug) that will be used for pulldown into each sample.</li>
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<ul><li>Antibody amounts can vary depending on the protein you pull down and the abundance of the protein in the lysates.</li></ul>
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<li>Add sepharose beads conjugated to <i>Staphylococcus aureus</i> protein A to the solution. Protein A binds to the Fc region of antibodies and will pull down the antibody bound to your protein. </li>
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<li>Incubate at 4 degrees for 2-4 hours on a rotator.</li>
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<li>Wash three times with TBST and spin at 5000 rpm for 5 minutes. </li>
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<li>Elute with 5x lamelli buffer with Beta- mercaptoethanol and boil at 96 C for 5 mins and run a western blot.</li> </ol>
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Revision as of 01:14, 28 October 2013

Immunoprecipitation

Materials:

  • Cell lysates
  • Protein A conjugated agarose beads
  • Antibody of interest:0.5-1ug/ reaction
  • lysis buffer: 50 mM NaH2PO4
300 mM NaCl
10 mM imidazole
Adjust pH to 8.0 using NaOH.
  • 0.5 mm glass beads
  • Rotator
  • All reagents to run a western blot
  1. Grow up overnight cultures in the correct selection marker.
  2. Spin down the pellets next morning and lyse the cells using bead beaters
    • Incubate sample with glass beads and lysis buffer containing 100uM protease inhibitor for 5 mins on ice and then shake for 5 minutes on a bead beater. Repeat twice.
  4. Do a Bradford assay to determine the protein concentration of your samples
  6. Combine 500ug-1000ug of each lysate
    • Amount of protein you add for each lysate will vary upon the abundance of your protein of interest in the lysate
  7. Add the antibody (0.5-1ug) that will be used for pulldown into each sample.
    • Antibody amounts can vary depending on the protein you pull down and the abundance of the protein in the lysates.
  8. Add sepharose beads conjugated to Staphylococcus aureus protein A to the solution. Protein A binds to the Fc region of antibodies and will pull down the antibody bound to your protein.
  9. Incubate at 4 degrees for 2-4 hours on a rotator.
  10. Wash three times with TBST and spin at 5000 rpm for 5 minutes.
  11. Elute with 5x lamelli buffer with Beta- mercaptoethanol and boil at 96 C for 5 mins and run a western blot.

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