Dominik Haudenschild, June 1993 at CBRC

• Use 50µg of antibodies, with 50µl of beads
- protein-A or Protein-G beads depending on what species and type of antibody used
- probably 5µg or 10µg is enough also, depends on type of antigen & affinity of antibody

• Incubate I° antibody with beads for 1 hour in ~0.5ml to 1ml PBS
- not longer

• Wash 3 times with PBS
- don’t vortex beads
- can use flat gel loading tip to remove all wash buffer from eppendorf tube without sucking up any beads, but do not dry beads

• If necessary, incubate protein-A/G beads without antibody and the protein to be immunoprecipitated to get rid of nonspecific binding to the beads, then discard or regenerate these beads

• Use 50µg to 500µg total protein in 0.5ml to 1ml PBS + 1% NP-40
- RIPA buffer if IP too dirty
- do for approximately 1 hour

• Wash 4 times
- first wash in same buffer as IP buffer to avoid precipitation
- three more washes in PBS + 1% NP-40 + 0.5M NaCl
- with last wash, bring beads into new eppendorf tube before aspirating wash
- can use flat gel loading tip to remove all wash buffer from bottom of eppendorf tube without sucking up any beads

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

Dominik Haudenschild (from Ken Dickerson)

• 50µl Protein-G Sepharose per reaction

• Wash 2 times with IP wash buffer
IP wash buffer is PBS, 0.1% Tween-20, 0.5M NaCl

• I° antibody if from whole sera or ascites:
- 990µl IP wash buffer + 10µl sera or ascites
- 2 hours room temperature on rocker

• I° antibody if from cell culture supernatant
- 500µl supernatant + 500µl IP wash buffer
- 2 hours room temperature on rocker

• Wash three times with IP buffer

• Incubate with labeled media O/N 4°C on rocker

• Wash four times with IP buffer

• Transfer beads to a new tube, boil in SDS-Page buffer, and run on gel.