(From Peter Baciu, June 1993)

• Use 50µg of antibodies, with 50µl of beads.

This level of Antibody is excessive but if results are needed you can start here. However, with monoclonals 1-3 µg should work. Before doing a number of experiments you should titrate out antibody to determine optimal antibody concentration. 5µg or 10µg is should be plenty. The exact concentration depends on type of antigen & affinity of antibody
- protein-A or Protein-G beads depending on what species and type of antibody used
Consult pierce catalog which gives the relative affinities for each antibody type.

• Incubate I° antibody with beads for 1 hour at room temperature in ~0.5ml to 1ml PBS to assure adequate mixing.
- not longer

• Wash 3 times with PBS or I.P. solution. It is best to use immunoprecipitation solution since this will also cut down on non-specific binding to beads.

- don’t vortex beads

• Use ~500µg cell lysate from ~106 cells per I.P.

-A good starting point is to use PBS+1% NP40, other detergents than NP40 can be used.

-Lysis cells on ice with detergent containing buffer for 20 min. then scrape and centrifuge for 30 min. in cold.

- pre-clear resulting supernatant with 100 µl of protein. G/A beads for 30 min. use pre-cleared supernatant for I.P.

-If problems with non-specific binding or protein solubility try RIPA buffer

- Incubate Antibody/beads for 1 hour at room temperature. Longer incubation times increases non-specific absorption to beads.

• Wash 4 times
- first wash in same buffer as I.P. buffer to avoid precipitation. Some proteoglycans and proteins will precipitate in 0.5M salt.

- three more washes in PBS + 1% NP-40 + 0.5M NaCl. Also RIPA buffer can be used as a wash. Some antigens don’t bind to Antibody well in RIPA , but the use of RIPA for washing will facilitate the removal of non-specifically absorbed proteins.

- with last wash, bring beads into new eppendorf tube before aspirating wash.

-remove all fluid from beads using a flat tip pipette tip.

• Boil beads in SDS-PAGE buffer, to run on gel
- usually run one third of I.P. on one gel lane