Team:Calgary/Notebook/Protocols/Immunoprecipitation

From 2013.igem.org

(Difference between revisions)
 
(3 intermediate revisions not shown)
Line 8: Line 8:
<h1>Immunoprecipitation</h1>
<h1>Immunoprecipitation</h1>
-
<p>Materials: </p>
+
<h2>Materials: </h2>
<ul> <li>Cell lysates</li>
<ul> <li>Cell lysates</li>
<li>Protein A conjugated agarose beads</li>
<li>Protein A conjugated agarose beads</li>
Line 16: Line 16:
<li>Rotator</li>
<li>Rotator</li>
<li>All reagents to run a western blot</li></ul>
<li>All reagents to run a western blot</li></ul>
 +
 +
<h2>Procedure</h2>
   
   
<ol> <li>Grow up overnight cultures in the correct selection marker. </li>
<ol> <li>Grow up overnight cultures in the correct selection marker. </li>
<li>Spin down the pellets next morning and lyse the cells using bead beaters</li>
<li>Spin down the pellets next morning and lyse the cells using bead beaters</li>
-
<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>
+
<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>Do a Bradford assay to determine the protein concentration of your samples<li>
+
<li>Do a Bradford assay to determine the protein concentration of your samples</li>
<li>Combine 500ug-1000ug of each lysate</li>
<li>Combine 500ug-1000ug of each lysate</li>
<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>
<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>
Line 28: Line 30:
<li>Incubate at 4 degrees for 2-4 hours on a rotator.</li>
<li>Incubate at 4 degrees for 2-4 hours on a rotator.</li>
<li>Wash three times with TBST and spin at 5000 rpm for 5 minutes. </li>
<li>Wash three times with TBST and spin at 5000 rpm for 5 minutes. </li>
-
<li>Elute with 5x lamelli buffer with Beta- mercaptoethanol and boil at 96 C for 5 mins and run a western blot.</li> </ol>
+
<li>Elute with 5x lamelli buffer with Beta- mercaptoethanol and boil at 96 C for 5 mins and run a <a href="https://2013.igem.org/Team:Calgary/Notebook/Protocols/WesternBlot"> western blot</a>.</li> </ol>
</section>
</section>
</html>
</html>

Latest revision as of 22:33, 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

Procedure

  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.
  3. Do a Bradford assay to determine the protein concentration of your samples
  4. 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
  5. 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.
  6. 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.
  7. Incubate at 4 degrees for 2-4 hours on a rotator.
  8. Wash three times with TBST and spin at 5000 rpm for 5 minutes.
  9. Elute with 5x lamelli buffer with Beta- mercaptoethanol and boil at 96 C for 5 mins and run a western blot.