Immunoprecipitation Protocol(生物信息站-生物医学实验方法学）

Immunoprecipitation Protocol

Materials and Buffers

Cell lysate; Protein A or Protein G slurry

Primary antibody

NP-40 Cell Lysis Buffer               Protease Inhibitor Cocktail (100X)

50 mM Tris-HCl pH 8.0           *   PMSF, 5 mg (50 µg/ml)

150 mM NaCl                           * Aprotinin, 100 µg (1 µg/ml)

1% NP-40                                 *   Leupeptin, 100 µg (1 µg/ml)

                                                  *   Pepstatin, 100 µg (1 µg/ml)

                                                  * 100% Ethanol bring up to 1 ml, aliquot and keep at minus 20°C.

Alternatively, buy Complete Mini, EDTA-free protease inhibitor cocktail tablets, 12211500 (11836170001), from Roche Diagnostics GmbH.

Instruments: Centrifuge; Rocking platform or rotator

Step I: Cell Lysate Preparation

Harvest approximately 107 cells.
Note: The total # of cells per ml and the cell equivalent loaded per lane of gel should be optimized specifically for each protein and antibody.

Wash cells with ~10 ml of PBS in a conical tube and spin at 400xg for 10 minutes.

Discard supernatant and repeat step 2.
Note: If using tissue culture dish adherent monolayer cells, wash cells in the flask two times with room temperature PBS and aspirate without disturbing the monolayer.

After the second wash, remove supernatant completely and resuspend the cell pellet in 1 ml of cold Lysis Buffer containing 1X Protease Inhibitor Cocktail (final concentration of 107 cells/ml). Gently vortex the tube.
Note: To prevent protease action effectively, the Lysis Buffer should be pre-chilled and all the following steps should be kept in the cold. The Protease Inhibitor Cocktail should be added to the required volume of the cold Lysis Buffer just before use.
Note: For adherent monolayers, add 1 ml of cold Lysis Buffer containing 1X Protease Inhibitor Cocktail per 100 mm culture dish.

Place the tube or the dish on ice for 30 minutes, with occasional mixing.

Spin cell lysate at 10,000xg for 15 minutes at 4°C.

Carefully collect supernatant, without disturbing the pellet and transfer to a clean tube. The cell lysate can be frozen at this point for long-term storage at minus 80°C. Discard pellet.

Step II: Cell Lysate Preclearing

Transfer 50 µl of the Protein G beads slurry (commercially available from several vendors) to an eppendorf tube and add 450 µl cold Lysis Buffer. Spin at 10000xg for 30 seconds and remove the Lysis Buffer. Wash one more time with 500 µl of cold Lysis Buffer. Resuspend the beads in 50 µl of cold Lysis Buffer.

Add this 50 µl of prepared Protein G slurry and 500 µl of Cell Lysate to an eppendorf tube and incubate on ice for 30-60 minutes.

Spin at 10000xg for 10 minutes at 4°C and transfer the supernatant to a fresh eppendorf. If any bead has been transferred, spin again and carefully transfer the supernatant to another fresh eppendorf tube.

Step III: Immunoprecipitation

Add 5-10 µg of antibody to the eppendorf tube containing the cold precleared lysate.
Note: This concentration of mAb is suggested as a starting point. Each investigator may desire to titrate the concentration of antibody and volume of cell lysate in preliminary experiments to extensively determine the optimal conditions.

Incubate at 4°C for 1 hour.

Add 50 µl of washed Protein G slurry in prechilled Lysis Buffer (prepared as instructed in Preclearing Step 1 above).

Incubate for 1 hour at 4°C on a rocking platform or a rotator.

Spin the eppendorf tube at 10000xg for 30 seconds at 4°C.

Carefully remove supernatant completely and wash the beads 3-5 times with 500 µl of Lysis Buffer. To minimize background, care should be given to remove the supernatant completely in these washes.

After the last wash, aspirate supernatant and add 50 µl of 1X Laemmli sample buffer to bead pellet. Vortex and heat to 90-100°C for 10 minutes.

Spin at 10000xg for 5 minutes, collect supernatant and load onto the gel.
Supernatant samples can be collected and kept frozen at this point if the gel is to be run later.

Follow manufacturer's instructions for SDS-PAGE. Stain the gel for visual analysis of the immunoprecipitated protein.
If using Western blot after this step, follow the accompanying Immunoblotting (WB) Protocol. TrueBlotTM is recommended for use as an anti-mouse IgG second step in Western blot. For more information about TrueBlotTM, visit the following web address:
http://www.ebioscience.com/ebioscience/whatsnew/trueblot.htm.

Preferably, the antibody used for the immunoprecipitation portion is not the same antibody used for the western blot portion. A different clone with the same specificity is recommended.
Another RIPA Recipe:
Tris-HCl: 50 mM, pH 7.4
NP-40: 1%
Na-deoxycholate: 0.25%
NaCl: 150 mM
EDTA: 1 mM
PMSF: 1 mM
Aprotinin, leupeptin, pepstatin: 1 microgram/ml each
Na3VO4: 1 mM
NaF: 1 mM
1. Add 790 mg Tris base to 75 ml distilled H2O. Add 900 mg NaCl and stir the solution until all solids are dissolved. Using HCl, adjust the pH to 7.4.
2. Add 10 ml of 10% NP-40 to the solution.
3. Add 2.5 ml of 10% Na-deoxycholate and stir until solution is clear.
4. Add 1 ml of 100 mM EDTA to the solution. Adjust the volume of the solution to 100 ml using a graduated cylinder. Store RIPA buffer at 2-8°C until ready to use.
5. Ideally, the remaining protease and phosphatase inhibitors should be added to the solution on the same day the assay is run (100 microliters of aprotinin, leupeptin, and pepstatin; 500 microliters of PMSF, Na3VO4, and NaF), but with the exception of PMSF the diluted inhibitors are stable in aqueous solution for up to 5 days. PMSF is extremely unstable in aqueous solutions with a half-life of approximately 30 minutes and should be added immediately before use, or instead of the inhibitors, you can try the commercial inhibitor-cocktails (Sigma and Roche).