Revision as of 01:14, 28 October 2013 by Wkeithvan (Talk | contribs)



  • 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.
  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.