Immunoprecipitation (IP) is the small-scale affinity purification of antigens using a specific antibody that is immobilized to a solid support such as magnetic particles or agarose resin. IP is one of the most widely used methods for isolating proteins and other biomolecules from cell or tissue lysates for the purpose of subsequent detection by western blotting and other assay techniques.

All Antibodies for use in IP

Considerations when choosing an IP antibody

  1. Whether it will work in IP. Not all antibodies work for all applications. It is important to ensure that the chosen antibody works in immunoprecipitation or another application that requires the target to be recognized in its native state. Other applications such as immunofluorescence (IF), immunocytochemistry (ICC), and immunohistochemistry (IHC) can be good indicators as to whether the antibody would work for IP, as well.
  2. Whether it is specific. Invitrogen antibodies undergo a two-part testing approach: functional application validation* and targeted specificity verification. Functional application validation provides information on whether the antibody works in IP. Target specificity verification ensures the antibody is recognizing the target protein of interest.
  3. Whether there is an antibody available for the target of interest. For proteins against which no antibodies are available yet, a suitable tag can be genetically introduced into the protein of interest. Then, the protein can be readily detected through tag antibodies.

Both monoclonal and polyclonal antibodies can work in IP. The key requirement is that the specific epitope of interest be exposed. One of the advantages of using a monoclonal antibody is that, generally, it is more specific. However, this is associated with a higher likelihood that the one epitope it recognizes is buried. Unless monoclonal antibodies are specifically screened or designed for use in IP, polyclonal and recombinant polyclonal antibodies are better candidates for recognizing target proteins, as they recognize multiple epitopes of the targets and allow for stable complexes to form between a polyclonal antibody and an antigen.

IP data examples

Each Invitrogen antibody that is indicated for IP applications has undergone functional application testing. Here are some examples of that testing.

Immunoprecipitation assay of endogenous PRMT3 protein using Anti-PRMT3 Antibody. PRMT3 was immunoprecipitated using 5 µg of the PRMT3 Recombinant Rabbit Monoclonal Antibody (Cat. No. 703744) from whole cell extracts (800 µg) of A549 (Lane 3) using the Protein A/G Dynabeads (Cat. No. 10001D and Cat. No. 10003D). Normal Rabbit IgG was used as an isotype control (Lane 2). Western blot analysis was performed using PRMT3 Recombinant Rabbit Monoclonal Antibody (Cat. No. 703744) at a 1:5000 dilution. The blot was detected by chemiluminescence. Known quantity of protein samples were electrophoresed using NuPAGE 10% Bis-Tris gel. Resolved proteins were then transferred onto a nitrocellulose membrane (Cat. No. IB23001) by iBlot 2 Dry Blotting System (Cat. No. IB21001). Chemiluminescent detection was performed using Novex ECL Chemiluminescent Substrate Reagent Kit (Cat. No. WP20005).

Immunoprecipitation assay of endogenous SIGLEC9 protein using Anti- SIGLEC9 Antibody. SIGLEC9 was immunoprecipitated using 5 µg of the SIGLEC9 Recombinant Rabbit Monoclonal Antibody (Cat. No. 703464) from whole cell extracts (200 µg) of THP-1 (Lane 4) using the Dynabeads Protein G Immunoprecipitation Kit (Cat. No. 10007D). Normal Rabbit IgG was used as an isotype control (Lane 3). Western blot analysis was performed using SIGLEC9 Recombinant Rabbit Monoclonal Antibody (Cat. No. 703464, 1:500 dilution) . A 55, 60 kDa band corresponding to isoforms of SIGLEC9 was observed in IP elute. The blot was detected by chemiluminescence. Known quantity of protein samples were electrophoresed using NuPAGE 10% Bis-Tris gel. Resolved proteins were then transferred onto a nitrocellulose membrane (Cat. No. IB23001) by iBlot 2 Dry Blotting System (Cat. No. IB21001). Chemiluminescent detection was performed using Novex ECL Chemiluminescent Substrate Reagent Kit (Cat. No. WP20005).

Resources

*The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.