Resources

Immunoprecipitation (IP) Protocol

Immunoprecipitation (IP) is one of the most widely used antibody-based techniques. It is used to purify and enrich the protein of interest from a complex mixture such as cell lysate, tissue homogenate or blood sample. The protein is captured by a specific antibody, and then the antibody-protein complexes are pulled out of the sample using Protein A/G-coupled agarose beads or magnetic beads. IP is an important step in many proteomics studies: the molecular weights of protein antigens, protein/protein interactions, post-translational modifications and expression profiling of proteins. The IP technique can also enable the detection of rare proteins which otherwise would be difficult to detect since they can be concentrated up to 10,000-fold by immunoprecipitation. Although researchers are increasingly choosing magnetic beads for IP purification, agarose-based IP remains a versatile and powerful option in many circumstances, especially where the need for scale-up is anticipated. Here we describe a detailed procedure for agarose-based IP to purify and enrich the protein of interest.

Reagents:

  • PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH to 7.4.
  • Cell lysis buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1mM EDTA, 1% NP-40, 1% Na-deoxycholate, 0.1% SDS, sterile-filtered, add protease inhibitor cocktail before use.
  • 3 × SDS buffer: 150mM Tris-HCl (pH6.8), 6%(W/V) SDS, 0.3%(W/V) BPB, 30% glycerol, 3% β-mercaptoethanol.
  • Glycine buffer: 0.10 M Glycine, 500 mM NaCl, 0.05M Tris-HCl (pH 1.5–2.5)
  • Tris-HCl: 1 M Tris, 4% (W/V) HCl, pH 8.0
  • Antibody

Equipment:

  • Refrigerated microcentrifuge
  • Sonicator
  • Protein A/G agarose beads

Procedures:

Preparation of cell Lysates

1. Collect the cells and spin at 500 × g for 5 min at 4°C. Remove the supernatant.

2. Wash cells with ice-cold PBS for three times. Spin at 500 × g for 5 min at 4°C and remove the supernatant.

3. Resuspend the washed cell pellet in ice-cold cell lysis buffer (1 mL per 1 × 107 cells) and incubate on ice for 10 min.

4. Sonicate cells three times for 5 second pulses each in ice bath to ensure the full release of the proteins from cells.

5. Pellet cellular debris by spin at 13,000 × g for 10 min and transfer the supernatant into a new tube. Store on ice for further use, or for long storage at -80°C.

Preparation of Protein A/G agarose beads

6. Wash the Protein A/G agarose beads twice with 1 mL PBS, centrifuge at 2,000 × g for seconds and remove the supernatant between washes.

7. Dilute beads 1:1 with PBS.

Note: It is recommended to use wide orifice pipette tips or tips with the end cut off when manipulating agarose beads to avoid disruption of the beads.

Immunoprecipitation

8. (Optional) Pre-clear the cell lysate: add 100 uL pre-washed Protein A/G agarose bead slurry per 1 mL of cell lysate and incubate at 4 °C for 10 min with gently agitation. Spin at 3,000 × g at 4 °C for 2 min, remove the beads and collect the supernatant in a new centrifuge tube

9. Add relevant antibody to the pre-cleared cell lysate. And gently rock the mixture at 4°C for 2-4 h or overnight.

Note: Optimal antibody concentration should be determined by titration.

10. Capture the immunocomplex by adding 25-40 uL pre-washed Protein A/G agarose bead slurry. Gently rock for 1 hour or overnight at 4°C.

11. Collect the beads by low speed centrifugation at 2,000 × g, 1 min at 4°C and remove the supernatant.

12. Wash the beads with cell lysis buffer (stringent) or PBS (less stringent), collect the beads by low speed centrifugation at 4°C and discard the supernatant.

13. Repeat Step 12 three times.

Elution

Protein can be eluted from the beads by denature buffer or non-denature buffer (low-pH glycine buffer). And increasing the salt concentration in elution buffer will help to elute protein. The eluent can be stored on ice for same day analysis or frozen at -80˚C for future analysis.

  • SDS buffer (denaturing) elution: resuspend the beads in 20 µL 3 × SDS buffer and boil for 5 min to dissociate the immunocomplex from the beads. Spin at 13,000 × g for 1 min at 4°C, and collect the supernatant in a new 1.5 mL Ep tube.
  • Low-pH (non-denaturing) elution: resuspend the beads in 40 µL glycine elution buffer and incubate for 10 min at room temptation with frequent agitation. Spin at 13,000 × g for 1 min at 4°C, and collect the supernatant in a new 1.5 mL Ep tube. Pool the eluate and neutralize by adding equal volume of Tris-HCl (pH 8.5). The beads can be reused after removal the glycine buffer.

Learn about the principle of IP.

If you have any question, please contact us at: .

Return to Resources

Inquiry Basket