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Team:Calgary/Notebook/Protocols/Immunoprecipitation
From 2013.igem.org
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<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> | ||
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| + | <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 | + | <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> | ||
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<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
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
- Grow up overnight cultures in the correct selection marker.
- 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.
- Do a Bradford assay to determine the protein concentration of your samples
- 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
- 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.
- 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.
- Incubate at 4 degrees for 2-4 hours on a rotator.
- Wash three times with TBST and spin at 5000 rpm for 5 minutes.
- Elute with 5x lamelli buffer with Beta- mercaptoethanol and boil at 96 C for 5 mins and run a western blot.
