Immunprecipitation with embryo extracts:




  • Collect staged embryos, dechorionate, determine the weight (1 mg corresponds to about 100 embryos, briefly spin and freeze in liq. N2, store at -80ºC
  • Perform the following steps at 4ºC. Optimally you work in the cold room and keep all the materials (e. g. pipette tips) and solutions in the cold room. Add PMSF and protease inhibitors to the lysis buffer.
  • Load the proteinA-sepharose beads with antibody. Wash the beads 3x with PBT. Spin gently, 1 min, 2k. Suspend the beads in 1 ml PBT and add the antibody/serum. Incubate for 1h on a rotating wheel. Spin gently, discard the supernatant and wash 3x with PBT.
  • Homogenise embryos with several (20-30) strokes in a Dounce homogeniser in lysis buffer (see below for buffer composition). Start with the "loose" pistle, then use the "tight" pistle. Lyse about 10 to 100 mg per 1 ml of lysis buffer. Alternatively, you may use the homogeniser with "Schliff" for a more complete lysis, if you do not want to separate the nuclei.
  • spin 15 min at 14k in 1,5 ml/2ml reaction tubes. Transfer the supernatant to a new tube. Be careful not to transfer the lipids floating on the extract. To remove lipids you may extract the lysate with 1 volume of Triflourtrichlorethan.
  • Add the clear lysate to 10 ul proteinA-sepharose beads loaded with antibody (about 1-5 ul of serum/1-5 ug antibody per 10 ul of beads)
  • Incubate for 1 h, rotate on a wheel. In case of instable proteins (e. g. slam incubate shorter (20 min).
  • spin gently (1 min, 2k), save the supernatant (unbound), and wash the beads 5x with washing buffer.
  • Elute the proteins with 50 ul of Laemmli buffer.

Optimisation:
- vary the amount of antibody/extract volume
- the antibody may be bound to the protein A prior to incubation with the extract
- IP buffer composition:  detergents, salt concentration
- clearing of the extract with protein A beads may reduce background binding


Materials

Protein A Sepharose (Pharmacia)


When pipetting the beads, cut off the tip of the yellow tip for a wider opening.
  • The beads are stored as dry powder in a dessicator. To prepare the slurry add 15 ml of water to 0,1 g powder (400-500 µl of gel) in a 15 ml falcon tube. Please be careful when opening the small container.
  • Spin 1 min at 2k, discard supernatant, repeat 2x
  • Suspend in small volume and transfer to an 1,5 ml vial
  • Store in 20% ethanol for several months at 4¡C
Please see the Product description for complete information.

Protease cocktail (Roche)

Dissolve one tablet in 10 ml of lysis buffer. You do not need to add protease inhibitors to the washing buffer. The tablet may also be dissolved in 1.5 ml of water as a 7X stock solution. Please refer to the product description for full information.

Buffers:


RIPA buffer dissolves membranes and in general results in low background. However due to the strong detergents it may disrupt protein-protein interactions and denature proteins. The IP buffer is more gentle and does not dissolve membranes. To all lysis buffer protease inhibitors should be added, including 1 mM PMSF, 1 mM EGTA, 1 mM EDTA, and the ROCHE protease inhibitor cocktail.

RIPA buffer:

    20 mM Tris/HCl pH 7.5
    150 mM NaCl
    0.1% SDS
    1% TritonX-100
    0.5% Deoxycholate

IP buffer

    50 mM Hepes/NaOH, pH7,5
    150 mM NaCl
    0,5% Triton X-100
    10% Glycerin

Washing buffer

    PBT (PBS + 0.1% Tween20)
    or
    20 mM Tris/HCl pH 7.5
    150 mM NaCl
    0.1% TritonX-100