Immunoprecipitation Using Cells Lysed With Detergent Under Denaturing Conditions
If epitopes of native proteins are not accessible to antibodies, or if the antigen cannot be extracted from the cell with nonionic detergents, cells should be solubilized under denaturing conditions. This protocol is based on that for nondenaturing conditions (see Basic Protocol 1, steps 1 to 7), with the following modifications. Denaturation is achieved by heating the cells in a denaturing lysis buffer that contains an ionic detergent such as SDS or Sarkosyl (N-lauroylsarcosine). The denaturing lysis buffer also contains DNase I to digest DNA released from the nucleus. Prior to immunoprecipitation, the denatured protein extract is diluted 10-fold with nondenaturing lysis buffer, which contains Triton X-100; this step protects the antigen-antibody interaction from interference by the ionic detergent. Immunoprecipitation is performed as described (see Basic Protocol 1).
The following protocol is described for cells in suspension culture, although it can be adapted for adherent cells (see Alternate Protocol 1). Only antibodies that react with denatured proteins can be used to immunoprecipitate proteins solubilized by this protocol.
Additional Materials (also see Basic Protocol 1) Denaturing lysis buffer (see recipe)
Heating block set at 95°C (Eppendorf Thermomixer 5436 or equivalent) 25-G needle attached to 1-ml syringe
1. Collect cells in suspension culture (see Basic Protocol 1, steps 1 to 3). Place tubes on ice.
2. Add 100 ^l denaturing lysis buffer per -0.5-2 x 107 cells in the pellet.
3. Resuspend the cells by vortexing vigorously 2 to 3 sec at maximum speed. Transfer suspension to a 1.5-ml conical microcentrifuge tube.
The suspension may be very viscous due to release of nuclear DNA.
Tubes can have flip-top or screw caps. Screw-capped tubes are preferred because they are less likely to open accidentally during subsequent procedures. They are also recommended for work with radioactivity.
4. Heat samples 5 min at 95 °C in a heating block.
5. Dilute the suspension with 0.9 ml nondenaturing lysis buffer. Mix gently.
The excess 1% Triton X-100 in the nondenaturing lysis buffer sequesters SDS into Triton X-100 micelles.
6. Shear DNA by passing the suspension five to ten times through a 25-G needle attached to a 1-ml syringe.
If the DNA is not digested by DNase I in the denaturing lysis buffer or thoroughly sheared mechanically, it will interfere with the separation of pellet and supernatant after centrifu-gation. Repeat mechanical disruption until the viscosity is reduced to manageable levels.
7. Incubate 5 min on ice.
8. Clear the lysate and perform immunoprecipitation (see Basic Protocol 1, steps 6 to 27).
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