Immunoprecipitation protocol
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O. Gjoerup 8-
1. If cells were transfected, harvest approximately 48 hrs after. Wash cells twice in PBS,
then scrape (cell scraper) into PBS, spin down, aspirate and extract cell pellet in 'an
appropriate lysis buffer'.
One frequently used buffer is based on NP40:
Buffer TEB: 50 mM Tris pH 7.5, 150 mM NaCl, 1.0 % Nonidet P-40, 10 % glycerol, 10
µg/ml leupeptin, 10 µg/ml pepstatin, 0.5 mM phenylmethylsulfonyl fluoride (PMSF).
Add the protease inhibitors immediately before use.
Use 1 ml TEB pr. 100 mm dish. Extract 15 min. on ice.
2. Spin in eppendorf centrifuge at top speed for 5-10 min (4 ˚C).
3. Transfer supernatant (lysate) to a new tube. Add antibody (often 1-2 µg for each I.P.;
translates into 2-3 µl polyclonal serum, or 50 µl hybridoma supernatant for monoclonals),
and allow binding of antibody to antigen for 2 hrs (4 ˚C).
4. Add 30 µl (approx. 50 % slurry) of protein A/G sepharose (e.g. Santa-Cruz) to the
lysate and incubate on a rotating device at 4 ˚C for an additional 45 min.
(If antibody is preconjugated you can just add the recommended volume of beads, around
30 µl I'd assume, directly to the lysate and incubate 45 -60 min.)
5. Washes. These are most conveniently done in a 'spinning bucket type' eppendorf
centrifuge at speed '3' 1 min. if this is possible. A regular eppendorf can also be used, but
aspirating without touching the beads may be more difficult. Don't spin beads to hard.
Aspirate with a fine pipet tip or a needle trimmed of at the end. Thus one can avoid
aspirating the beads accidentally. Do 3-5 washes with your lysis buffer, e.g. 1 ml each
time and mixing up the beads each time. If you want to wash more stringently, e.g. high
salt, do two washes with 0.5 M LiCl and finish off with a lysis buffer wash to get rid of
the salt.
6. Aspirate the last wash from the beads, add an appropriate volume of SDS sample
buffer with reducing agent and boil 3-5 min. Often 50-60 µl is good for a large gel. Spin
down right before loading to avoid getting beads into the pipet tip.
Remember: When doing I.P.- blots you will get a blasting heavy and light chain signal
(55 kDa and 25 kDa) if each of the two antibodies used for I.P. and blot are from the
same species. Minor bands will show up if they're different species.