Immunoprecipitation with Resins

Immunoprecipitation (IP) is the small-scale affinity purification of antigens using a specific antibody. IP enables researchers to enrich for low-abundance proteins in order to improve downstream analysis, such as identifying the activation status, determining post-translational modifications, or capturing protein-binding partners (co-immunoprecipitation, co-IP). Although researchers are increasingly choosing magnetic beads for IP-scale purification, agarose-based IP remains a versatile and powerful option in many circumstances, especially where the need for scale-up is anticipated.

Find magnetic bead- and agarose bead-based kits using immunoaffinity or posttranslational modification–specific affinity binding to achieve protein enrichment and cleanup prior to mass spectrometry.

In addition to Chromatin Immunoprecipitation Assays (ChIP), Thermo Scientific offers complete and easy to use DNA and RNA Electrophoretic Mobility Shift Assay (EMSA) Kits, to study the binding of a protein to a known DNA or RNA oligonucleotide probe to assess the affinity of the interaction.

Select from resin- and filter-based columns and kits to enrich or purify protein samples for mass spectrometry analysis. Techniques include immunoprecipitation, enrichment by PTM (such as phosphoprotein enrichment), or active site labeling and enrichment using activity based probes.

Immunoprecipitations were performed according to the product instructions using 10 µg of affinity-purified goat anti-GFP antibody and the Pierce Direct, Classic and Crosslink IP Kits. The cell lysate was prepared using IP Lysis/Wash Buffer and pre-cleared using the Pierce Control Agarose Resin supplied in the kits. The immune complex was formed by incubating the antibody, resin, and lysate overnight. The resin was washed with IP Lysis/Wash Buffer, 1X Conditioning Buffer and eluted with Elution Buffer. For analysis, 4-20% tris-glycine gels were loaded with 20% of the eluted sample, 5% of the cell lysate load (Lysate) and 10% of the antibody load (IgG) and stained with Imperial Protein Stain. For the resin controls, the immunoprecipitation was performed without adding the antibody. MW: molecular weight markers.

A431 lung carcinoma cells were cultured in DMEM containing 10% FBS for 24 h. Following a 24 h serum withdrawal, half of the cultures plated were treated with 100 ng/mL EGF for 10 min. Crosslinking was achieved using 1% formaldehyde in the media for 10 min. ChIP assays were performed according to the manufacturers’ protocols. Quantitative real-time PCR data was obtained with a Bio-Rad iQ5 Thermocycler, ABsolute QPCR SYBR Green Fluorescein Master Mix, and primers designed to amplify a region of the human MYC promoter proximal to the transcription start site.

HA-tagged pRb was expressed using a Pierce Human in vitro Expression System. 50 µL of the translation reaction was diluted to 200 µL in TBS and added to 15 µL of agarose resin and incubated for 1 h at 4°C as a non-specific pre-clearing step. The cleared translation reaction was added to 50 µL of anti-HA resin and incubated for 3 h at 4°C with end-over-end rocking. The resin was washed with 3 x 500 µL of TBS-T. HA-pRb:E2F1 complex was eluted with the addition of 30 µL of 2X non-reducing sample buffer. In vitro translation reactions without and with HA-pRb DNA (lanes 1 and 3, respectively) and anti-HA agarose elution fractions from each (lanes 2 and 4, respectively) were probed with an rabbit anti-E2F1 and mouse anti-HA and detected with Thermo Scientific SuperSignal West Dura Extended Duration Substrate.