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.
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.