Mini Protein Extraction/Co-Immunoprecipitation Assay

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  1. Grow an overnight culture in 5ml of liquid medium (Vogel's N + Sucrose)
  2. Harvest mycelium using a buchner funnel, vacuum flask, plus a #4 Whatman paper disk
  3. Wash mycelium with ~ 100mls of H2O or PBS buffer
  4. Trasfer washed mycelium to a 1.5ml epi-tube containing 500ul Protein extraction buffer
  5. Sonicate for 30 1-second pulses (Output 3.5, duty cycle 80)
  6. Centrifuge @ 4 degrees C for 5 min to pellet debris (14,000rpm)
  7. Transfer supernatant to a new, labeled epi-tube.
  8. For analysis of crude extracts or for saving an input sample for CoIP experiments, transfer an aliquot of the crude extract to a epi-tube containing the appropriate volume of 4X SDS loading buffer, boil for 3 minutes, and store in the -20 freezer until ready to use.
  9. For CoIP experiments, mix the remaining crude extract with the desired antibody or antibody resin and incubate overnight on a rotator in the cold room. (I typically add 1ul of antibody to 300ul of crude extract in an epi-tube.)
  10. The next morning add 20ul of Protein A/G beads that have been equilibrated in extraction buffer (see instruction below). Incubate for 2 hr on a rotator at 4 degrees C.
  11. Wash the agarose beads 2X's with 1ml extraction buffer.
    1. Pellet the beads by spinning for 1 min at 5000rpm in the cold room.
    2. Remove the supernatent (containing unbound proteins) with a pipette.
    3. Add 1ml of Protein Extraction Buffer
    4. Invert tube several times to suspend beads.
    5. Repeat
  12. Pellet the beads and remove the supernatent.
  13. Add 40ul of 2X SDS loading buffer to each sample, boil for 3 minutes, and store in the freezer until ready to use.

Protein Extraction Bufer (10mls)

.5ml 1M HEPES buffer pH7.5
0.375mls 4M NaCl
20ul 0.5M EDTA
20ul 10% NP-40
50ul 0.2M PMSF (be careful!)
1 tablet Roche complete mini protease inhibitor cocktail
9.085mls H2O

Equilibration of Protein A/G Beads

  1. Determine the amount of beads you need for all your reactions (To make sure I have enough beads, I equilibrate 30ul for each IP sample I plan to do, but I only use 20ul in the actual IP.)
  2. Cut the end off a pipette tip to provide a wider opening for pipetting the beads.
  3. Pipette the total amount of beads into two epi-tubes. (e.g. if I need to equilibrate beads for 4 samples, I pipette 60ul of beads each into two epitube - 120ul beads total.)
  4. Add 1ml of cold Protein Extraction Buffer to each tube.
  5. Invert to mix and pellet beads at 5000rpm. (room temp centrifuge is o.k., but keep beads on ice while working with them)
  6. Pipette off supernatent.
  7. Repeat steps 4-6 two more times.
  8. Add enough protein extraction volume to restore the original volume.
  9. Add 20ul of beads to each tube containing extract and antibody.