Mini Protein Extraction/Co-Immunoprecipitation Assay
From Lewis Lab Wiki
- Grow an overnight culture in 5ml of liquid medium (Vogel's N + Sucrose)
- Harvest mycelium using a buchner funnel, vacuum flask, plus a #4 Whatman paper disk
- Wash mycelium with ~ 100mls of H2O or PBS buffer
- Trasfer washed mycelium to a 1.5ml epi-tube containing 500ul Protein extraction buffer
- Sonicate for 30 1-second pulses (Output 3.5, duty cycle 80)
- Centrifuge @ 4 degrees C for 5 min to pellet debris (14,000rpm)
- Transfer supernatant to a new, labeled epi-tube.
- 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.
- 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.)
- 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.
- Wash the agarose beads 2X's with 1ml extraction buffer.
- Pellet the beads by spinning for 1 min at 5000rpm in the cold room.
- Remove the supernatent (containing unbound proteins) with a pipette.
- Add 1ml of Protein Extraction Buffer
- Invert tube several times to suspend beads.
- Pellet the beads and remove the supernatent.
- Add 40ul of 2X SDS loading buffer to each sample, boil for 3 minutes, and store in the freezer until ready to use.
|.5ml||1M HEPES buffer pH7.5|
|50ul||0.2M PMSF (be careful!)|
|1 tablet||Roche complete mini protease inhibitor cocktail|
Equilibration of Protein A/G Beads
- 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.)
- Cut the end off a pipette tip to provide a wider opening for pipetting the beads.
- 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.)
- Add 1ml of cold Protein Extraction Buffer to each tube.
- Invert to mix and pellet beads at 5000rpm. (room temp centrifuge is o.k., but keep beads on ice while working with them)
- Pipette off supernatent.
- Repeat steps 4-6 two more times.
- Add enough protein extraction volume to restore the original volume.
- Add 20ul of beads to each tube containing extract and antibody.