Protein Protocols Protein Protocols

1104 Johansen and Svensson
4. If the antigen of interest is a secreted antigen, the radioactive supernatant is saved for
immunoprecipitation. If the antigen of interest accumulates intracellularly, the radioactive
supernatant is discarded in the radioactive waste.
3.2. Lysis of Cells
1. Wash the cells twice with ice cold PBS containing 20 mM NEM, drain the last PBS with a
Pasteur pipet and add the lysis buffer of choice (2–3 mL to a 25-cm2 flask) (see Note 3).
Let monolayers solubilize for 30 min on ice.
2. To clear the lysate from cell debris, centrifuge for 10 min at 12,000g in a microfuge.
Before storage of the labeled antigen at –70°C, aliquot the antigen to avoid repeated freezing
and thawing (see Note 4).
3. Check efficiency of metabolic labeling by running an SDS-PAGE and autoradiography.
3.3. Formation of Antigen–Antibody Complexes
1. Mix appropriate amounts of the cell lysate (usually 5–100 µL) with the monoclonal or
polyclonal antibody and dilute to 500 µL in RIPA buffer and incubate at 4°C overnight. Aim
at a sufficient amount of antibody to precipitate all of the target antigen. Several factors,
such as concentration of antigen and titer and avidity of the antibody, will affect the amount
of antibody to be used. Start the immunoprecipitation by titrating the antibody against a
fixed amount of target antigen.
Complete immunoprecipitation is usually obtained by 0.5 µL–5 µL of polyclonal
antiserum, 5–100 µL of hybridoma tissue culture medium, or 0.1–1.0 µL of ascitic fluid
(see Note 6).
2. If your antibody does not bind efficiently to protein A, G, or jacalin, add a second antibody
that is directed against your primary antibody and binds strongly to one of these
proteins, and incubate at 4°C for 1 h. The amount of anti-immunoglobulin antibody must
be titrated against a fixed amount of antigen–primary antibody complex to exclude reactivity
between secondary antibody and target antigen.
3. Add 25–100 µL of protein A-/protein G-Sepharose beads or jacalin-agarose beads to the
antigen–antibody mixture and incubate on a rocker for 1 h at 4°C or at room temperature.
4. Centrifuge the newly formed immune complexes at 12,000g for 30 s and remove the
supernatant. Wash the complexes to remove nonspecifically adsorbed proteins at least
four times with 1 mL of RIPA buffer and resuspend the beads with careful vortexing
between washes. The last wash should always be performed with the low-salt washing
buffer. Take care to remove the last traces of the final wash.
5. To disassociate the immune complexes, add 40 µL of sample buffer and incubate at 100°C
for 2–3 min (see Note 7). Centrifuge the samples for 20 s at 12,000g in a microfuge and
save the supernatants for SDS-PAGE. Samples can be frozen at –20°C before analysis
by SDS-PAGE.
3.4. SDS-PAGE
Prepare an SDS-PAGE gel as described in Chapter 11, Subheading 3. Following
the run, fix the gel.
3.5. Detection and Analysis of Immunoprecipitated Proteins
Autoradiography:
1. After washing the fixed gel twice in deionized water soak the gel in 50–100 mL of 1 M
sodium salicylate in deionized water for 30 min on a rocker (see Note 10).