Agarose Immunoprecipitation Kits

We offer a variety of kits for immunoprecipitation (IP) and co-immunoprecipitation (co-IP) that utilize different methods to capture the antibody against the target protein (and in some cases, interacting proteins) of interest. These methods utilize immobilized Protein A/G or activated supports (Aminolink or Glycolink) for direct antibody immobilization to agarose-based beads.

• More choices—different strategies to accommodate various antibody and downstream analysis needs
• Effective—require minimal antibody (2–10 μg) and are highly efficient in capturing antigens
• Complete—include all lysis, binding, wash, and elution buffers, with co-IP kit containing positive control lysates
• Fast—contain spin columns and collection tubes that shorten the protocol and minimize sample handling
• Flexible—can scale up or down as needed using adaptable protocols

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.

E. coli cells expressing fusion of green fluorescent protein (GFP) were extracted with B-PER Bacterial Protein Extraction Reagent and then immunoprecipitated using a polyclonal goat anti-GFP antibody. Eluted IP products were separated by SDS-PAGE and stained with the Coomassie stain GelCode Blue Stain Reagent to detect total protein. Lane 1 shows the single product obtained using the Crosslink IP Kit. Lane 2 displays the multiple antigen and antibody fragments obtained using a traditional IP method. MW is a molecular weight marker.

Purified rabbit polyclonal anti-STAT1 or rabbit polyclonal anti-EGFR was oxidized and coupled to 20 µL of resin. A549 cell lysate (0.5 mg) was incubated with the coupled resin for 1 hour at room temperature. Eluted proteins were resolved on a 4–20% SDS-PAGE Gel, transferred to a nitrocellulose membrane, and probed with the appropriate antibodies. Detection was performed using SuperSignal West Dura Chemiluminescent Substrate and a CCD-Imager (10-second exposure).