Immunoprecipitation technology

Immunoprecipitation (IP) is one of the most widely used immunochemical techniques. Immunoprecipitation followed by SDS-PAGE and immunoblotting, is routinely used in a variety of applications: to determine the molecular weights of protein antigens, to study protein/protein interactions, to determine specific enzymatic activity, to monitor protein post-translational modifications and to determine the presence and quantity of proteins. The IP technique also enables the detection of rare proteins which otherwise would be difficult to detect since they can be concentrated up to 10,000-fold by immunoprecipitation.

In the IP method, the protein from the cell or tissue homogenate is precipitated in an appropriate lysis buffer by means of an immune complex which includes the antigen (protein), primary antibody and Protein A-, G-, or L-agarose conjugate or a secondary antibody-agarose conjugate. The choice of agarose conjugate depends on the species origin and isotype of the primary antibody. The methods described are comparable and the choice of method depends on the specific antigen-antibody system.

REAGENTS AND EQUIPMENT

1. Agarose conjugate: fixed on Sepharose ® A protein on CL-4B (product number P405880) or G protein agarose 4B (product number R3944624) or L protein agarose (product number P394623), or an antibody agarose conjugate (based on the species origin of the first antibody and the homotype of the second antibody conjugate)

2. Preparation of cell or tissue lysates

3. Immunoprecipitation primary antibody and non related antibody (negative control)

4. SDS-PAGE and immunoblotting reagents and equipment

5. HNTG buffer: 20mM HEPES buffer, pH 7.5 (product number H109406), containing 150mM NaCl (product number S301560), 0.1% (w/v) Triton X-100 (product number T434565), and 10% (w/v) glycerol (product number G424796)

6. Washing buffer (cold): HNTG buffer, or PBS pH 7.4 (product number R355028), or other buffer (RIPA bufferproduct number R488105) depending on the required washing severity

7. Lyme sample buffer with or without 2-mercaptoethanol (2-ME (product number M301574) (such as 1x, 2x, or 3x)

8. Micro centrifugal tube

9. Micro centrifuge and vibrating screen

PROCEDURE

Immunoprecipitation of Specific Antigens

Note: Perform all IP steps using microcentrifuge tubes on ice unless noted otherwise.

Wash agarose conjugate twice with washing buffer, centrifuge for 10 sec. at 12,000xg at room temperature. Discard supernatant.

Note: If agarose conjugate is a powder, reconstitute it with deionized H2O and allow it to swell for 5 minutes.

Resuspend agarose conjugate in washing buffer (50% suspension).

Continue with method A or Method B, as desired.

Method A

1. Divide agarose conjugate into aliquots of 50-100 µL (approx. 25-50 µL agarose/bed volume) in microcentrifuge tubes.

2. Add to each tube 10 µL of primary antibody at appropriate dilution

3. Incubate for 15-60 min at room temperature, gently mixing the sample on a suitable shaker.

4. Centrifuge at 3,000xg for 2 min. at 4 °C. Discard supernatant.

5. Wash samples each with 1 mL washing buffer, centrifuge at 3,000xg for 2 min. at 4 °C. Repeat this step at least twice.

6. Add to each tube 0.1-1.0 mL of cell lysate.

7. Incubate for 90 min. to overnight at 4 °C, gently mixing the sample on a suitable shaker.

8. Collect immunoprecipitated complexes by centrifugation at 3,000xg for 2 min. at 4 °C. Discard supernatant.

9. Wash pellet with 1 mL washing buffer, centrifuge at 3,000xg for 2 min. at 4 °C. Repeat this step at least 3 times.

Method B

1. Add to cell lysate sample (0.1-1.0 mL), 10 µL of antibody at appropriate dilution (refer to product specification).

2. Incubate for 90 min. to overnight at 4 °C, gently mixing the sample on a suitable shaker.

3. Add 50-100 µL of agarose conjugate suspension (approx. 25-50 µL agarose/bed volume).

4. Incubate for 15-60 min. at 4 °C, gently mixing the sample with a shaker.

5. Collect immunoprecipitated complexes by centrifugation at 3,000xg for 2 min. at 4 °C. Discard supernatant.

6. Wash pellet with 1 ml washing buffer by resuspension and centrifugation at 3,000xg for 2 min. at 4 °C. Repeat this step at least 3 times.

Preparation for SDS-PAGE

1. Resuspend each pellet in 25-100 µL Laemmli sample buffer to a final concentration of 1x sample buffer. Heat samples at 95 °C for 5 min.

2. Centrifuge for 30 sec. at 12,000xg at room temperature. Collect supernatant (IP sample). If required, (and where protein stability permits) IP samples can be stored in sample buffer at -70 °C.

3. Run samples and MW standards with known concentrations on SDS-PAGE (appropriate percentage of polyacrylamide gel is according to the molecular size of the protein).

4. Transfer to nitrocellulose and perform immunoblotting.

Frequently Asked Questions and Answers about Immunoprecipitation (IP)

Can antibodies used for general immunization be used for immunoprecipitation experiments?

Answer: The nature of the antibody has a significant impact on the immunoprecipitation experiment. Different antibodies have different binding abilities to antigens and Protein G or Protein A. Therefore, antibodies that are generally immune to binding may not be able to be used for IP reactions. Generally speaking, polyantibodies are the best choice for precipitation reactions. In addition, purified monoclonal antibodies, ascites, and hybridoma supernatants can also be used for immunoprecipitation.

Why should a certain amount of protease inhibitors be added to the buffer for dissolving antigens?

Answer: Samples derived from the lysis of active cells often contain a certain amount of protease. In order to prevent the decomposition and modification of pre detected proteins, the buffer for dissolving antigens must be added with protein per inhibitor and tested at low temperature.

What should be paid attention to in the preparation of buffer solution for dissolving antigens?

Answer: Most antigens are cellular proteins, especially skeletal proteins, which must be dissolved in a buffer solution. For this reason, it is necessary to use a buffer containing a strong surfactant, although it may affect the binding of some antigens and antibodies. On the other hand, if cells are dissolved with weak surfactants, they cannot fully dissolve cellular proteins. Even dissolution produces binding results with other proteins, and the antigenic determinant is blocked, affecting binding to antibodies.

How should the ratio of antibody to buffer be grasped in immunoprecipitation experiments?

Answer: It is important to consider the ratio of antibody/buffer before each precipitation experiment. "If there are too few antibodies, the antigen cannot be detected, and if there are too many antibodies, they cannot settle on the beans, leaving a residue in the supernatant."; Too little buffer can not dissolve the antigen, while too much buffer can dilute the antigen. The specific proportion depends on the specific situation.

How should immunoprecipitation prepare cell lysates?

Answer: The choice of an appropriate cell lysate depends on the characteristics of the protein being studied. Triton X-100 and NP40 are non ionic interfacial active agents with mild effects; DOC (sodium deoxycholate), SDS, is a strong ionic surfactant. Therefore, the conditions such as the type and concentration of detergent used in the preparation of pyrolysis liquid, as well as the salt concentration, need to be optimized by the experimenter. Note that if you forget to add Triton X-100 and only add DOC or SDS, it may completely inactivate the antibody.

Reference

[1] Harlow E, Lane D. 1988. Antibodies: A Laboratory Manual. Laboratory Press New York: Cold Spring Harbor.