Immunoprecipitation

Outline - Supplies / Equipment - Reagents - Procedure 1 - Procedure 2 - Time required -

Outline

Immunoprecipitation is a technique that permits the purification of specific proteins for which a antibody has been raised. This primary antibody is either already bound to agarose or can be bound to the protein A/agarose beads during the procedure in order to physically separate the antibody-antigen complex from the remaining sample.

Supplies / Equipment

• sterile 1.5 µl microfuge tubes
• sterile pipette tips
• micropipettes
• waterbath 100°C
• microfuge 4°C
• rocking platform 4°C
• rocking platform 25°C / room temperature

Reagents

• Immunoprecipitation dilution buffer
• Immunoprecipitation wash buffer
• Immunoprecipitation buffer (cell lysis buffer 2)
• Electrophoresis sample buffer
• Antisera 0.5 - 5 µl, antibody-agarose conjugates
• 10% SDS

Procedure 1 (from Transduction Laboratories, Inc.)

1. Preparation of cell lysate (denaturing conditions)
   1. Rinse the cells on a confluent 60mm culture dish with PBS,
   2. Lyse with the addition of 0.5ml boiling 1% SDS, 1.0mM sodium vanadate, 10mM Tris-HCl pH 7.4.
   3. Transfer lysate to a 1.5ml microcentrifuge tube and boil for an additional 5 minutes.
   4. Sonicate briefly or pass several times through a 26 gauge needle and centrifuge for 5 minutes. This is a "total cell lysate (denatured)".
2. Preparation of cell lysate (native conditions)
   1. Rinse the cells in a confluent 60 mm culture dish with PBS, then lyse the cells with the addition of 0.5 ml cold immunoprecipitation buffer (cell lysis buffer 2), maintaining constant agitation for 30 minutes at 4°C.
   2. Scrape the cells from the dish and pass several times through a 26 gauge needle to disperse any large aggregates.
   3. Remove insoluble materials by centrifuging the cell lysates for 15 minutes at 4°C in a microcntrifuge. The supernatant is the "total cell lysate (native)".
3. Immunoprecipitation with soluble antibodies
   1. To a microcentrifuge tube, add 1-5 µg polyclonal or monoclonal antibody, 400 µl dH₂O, 500 µl of 2 x immunoprecipitation buffer, and 100 µl total lysate containing approximately 200 - 500 µg total protein.
   2. Vortex and incubate at 4°C for 1 hour. (If monoclonal antibodies are used, add 5 µg rabbit anti-mouse IgG antibody, vortex , and continue the incubation for an additional 30 minutes at 4°C).
   3. Add 50 µl 10 % protein A (S. aureus, Cowan strain) or protein A-sepharose. Vortex and incubate with agitation for 30 minutes at 4°C.
   4. Wash 3 x by centrifugation (spin 4 min. in a microfuge) with 1 x immunoprecipitation buffer.
   5. Resuspend the pellet in 30 µl of 2 x concentrated electrophoresis sample buffer, boil for 5 min., the centrifuge for 5 min.
   6. Load the supernatant onto an SDS-PAGE gel and electrophorese normally. Transfer to PVDF and probe with appropriate antibodies.
4. Immunoprecipitation with Antibody-Agarose Conjugates
   1. To a microcentrifuge tube, add 25µl of a 50% suspension of antibody-agarose conjugate, 400µl H2O, 500µl 2X immunoprecipitation buffer, and 100µl total lysate containing 200-500µg total protein.
   2. Vortex and incubate at 4°C for 1 hour.
   3. Wash with 1X immunoprecipitation buffer by centrifuging for 4 minutes in a microcentrifuge. Repeat wash.
   4. Resuspend the pellet in 30µl 2X concentrated electrophoresis sample buffer, boil for 5 minutes, then centrifuge for 5 minutes.
   5. Load the supernatant onto an SDS-PAGE gel and electrophorese normally. Transfer to PVDF and probe with appropriate antibodies.

Procedure 2 (from Chris Cohick)

Immunoprecipitation of a serum sample

1. Add 1/10 vol. of 10% SDS to sample. Vortex and heat for 4 min. at 100°C.
2. Immediately add 4 volumes of immunoprecipitation dilution buffer. Vortex and spin 5 min. in microfuge.
3. Transfer to a new tube, avoiding pellet.
4. To a microfuge tube, add 1-5 µg of primary antibody. Place on 4 °C rocker overnight.
5. If primary antibody was not already conjugated with agarose, add 20 µl protein A-sepharose per tube.
6. Place on 25°C rocker for 90 min., spin down pellet (5 seconds high speed).
7. Wash pellet 3 times with detergent wash buffer, then once with non-detergent wash buffer.
8. Add treatment buffer. Add 1/20 volume 2-mercaptoethanol.
9. Boil samples 4 min. Spin down pellet. Remove and save supernatant, this is the sample.

Time required

Afternoon and next morning.