Immunoprecipitation using C. elegans lysates

M. Soto, 3/2004

  1. Planning the IPs
    • Calculate what volume of packed beads you will need.
    • Figure out if you need Prot. A beads, Prot. G, or a mix (depends on 1ary antibody).
    • Prepare Lysis Buffer with DTT and detergent, (protease inhibitors optional).
    • Plan how much total lysates you will use.
    • Thaw frozen lysates on ice. Keep on ice at all times except when working in the cold room.
  2. Pre-clear to remove non-specific binding to the beads:
    Use a cut-off pipette tip to pick up beads needed for the experiments. Spin down the beads (2 min. at 3K or short quick spin), remove storage buffer (usually stored in alcohols). Rinse the beads in Lysis Buffer. Spin down. Repeat rinse in Lysis Buffer two more times.
    * Optional Control: save 1/10th volume of the lysates to be run on the gel as “starting reagent”.
    Add 20µl of beads, now in Lysis Buffer, to each lysate. Place on nutator in cold room for 30 min. to 1 hour. Spin down (3K for 5 min.). SAVE SUPERNATANT. Transfer to a fresh Epp. tube, pre-labeled, pre-cooled on ice.
    * Optional Control: save the Pre-Clear beads to run on a gel to see which non-specific bands you get.
  3. Primary antibody and immunoprecipitation
    Bring the lysates up to the desired amount using Lysis Buffer, at least 100µl. Record this volume in your notes. Add the recommended amount of primary antibody to the lysates. Put on nutator at 4 degrees for 30 min. Then add the Protein A or Prot. G beads (which are already in Lysis Buffer, not storage buffer) to the lysates for one to two hours. Amount of beads: minimum is 10µl, you can use more, e.g. 20µl. The beads have a “binding capacity” so over a certain amount, you are wasting beads, but you need enough so that you can easily spin them down.
  4. Washes
    * Optional Control: Save the supernatant to see how much of your protein is NOT binding to the beads. Run as much as you can fit in a lane (e.g. 30 µl/150µl) on the western.
    Remove the supernatant. Save it to a labeled Epp. tube if you want to check it (see above).
    Add 100µl Lysis Buffer, mix by inversion, spin down beads (2,5K for 2 min). Remove supernatant, repeat. Varietions: you can do the 3 washes for 5 min. each on nutator. Do a total of 3 washes.
    Remove the supernantant. The next step depends on what you are doing with the IPs.
    • Kinase Assays: do one wash in Kinase Buffer, continue.
    • Westerns of IPs: resuspend the beads in 1X Sample Buffer. Figure out what volume is ideal for later loading your gel. Add 2X Sample Buffer to the control beginning lysates.
    • Biorad/Hoeffer: now boil the sample for 5 min, spin down quickly, load supernant on gel.
    • Invitrogen: follow manufacture’s instructions.

Recording your experiments

Here is a template for how to record what you are loading on each gel. Excel files with the same information works well too. Make sure to date all of your experiments.

  • Gel #1: 10 % Tris-CL gel, poured fresh, ran at 120 V for 2 hours; lysates from 3/3/04.
  • Lysate amounts: Thawed 100µl of 10mg/ml = 1mg total. Lane 1 is 1/10 = 100µg.
  • Split remaining 900µg into three samples, tested Sigma Prot A, Amersham Prot A, and Amersham A+G Mix on 300µg of lysate. Brought each lysate up to 150 µl volume.
  • Lysis Bufffer has 0.1% Triton.
Lanes 1 2 3 4 5 6 7 8 9 10
Lysates N2 N2 N2 N2 N2 N2 N2 N2 N2 M
Starting amount 100 µg 300 µg     300 µg     300 µg    
Pre-clear * none A-S* A-S* A-S* A-A* A-A* A-A* A/G* A/G*  
IP none - CDK-1 >   >   >   >
Blot (Ab) PST Ab. 1:200   >   >   >    
Secondary anti-Rabbit HRP 1:2,000              
Notes Lys. PC IP Sup. PC IP Sup. IP Sup.  
* Prot A beads from SIGMA, A-S, and Amersham, A-A, A/G = Amersham A + G.