Immunoprecipitate multiple samples using a 96-well microplate format.

Thermo Scientific Pierce Coated Plate IP Kits provide for rapid immunoprecipitation of multiple samples without the usual tedium of pipetting, centrifuging and separating beaded affinity resin in individual microcentrifuge tubes. Immunoprecipitation is accomplished using coated 96-well microplates rather than beaded agarose resin. The plate format allows for faster processing of multiple samples. Select from Protein A/G or streptavidin coated plates.

1. Prepare pre-coated Protein G plate.     Wash 3 x 200 µl with IP Buffer.	5. Wash plate wells.     Wash 5 x 200 µl with IP Buffer.
2. Bind capture antibody to plate wells.     Incubate for 1 hour at room temperature.	6. Elute target antigen and capture antibody.     Add 50-100 µl Elution Buffer. Incubate 10-15 seconds.
3. Wash plate wells.     Wash 5 x 200 µl with IP Buffer.	7. Using a multichannel pipettor, transfer eluted samples    to an uncoated plate containing neutralization buffer.
4. Add antigen to appropriate test and control wells.     Incubate for 1 hour at room temperature.	8. Prepare samples for gel analysis.     Cover plate. Incubate for 5-10 minutes at 95°C.     Prepare for gel electrophoresis (reducing and     nonreducing conditions).

Immunoprecipitation of CD71 (transferring receptor) from human serum using Thermo Scientific Pierce Protein A/G Coated Plate IP Kit (Product # 45350) and a goat anti-CD71 polyclonal antibody. Eluted products for the experimental and control samples were mixed with nonreducing sample loading buffer, separated by SDS-PAGE, transferred to nitrocellulose membrane and detected by Western blotting with the IP antibody, Goat-anti-mouse-HRP conjugated secondary antibody (Product # 31432) and SuperSignal West Dura Chemiluminescent Substrate (Product # 34076). Lane 1: Experiment (immunoprecipitation product) Lane 2: Antibody only control (no sample) Lane 3: Human serum sample control (no antibody) Lane 4: Plate control (no antibody or human sample) Lane 5: Pure target protein (CD71) for size reference

Highlights:

• Ready-to-use, high quality coated plates provide high capacity and consistency
• Plate format best suited for simultaneously processing multiple samples and their control conditions
• Faster, easier and more thorough washing than with traditional tube/resin IP methods
• Uses familiar and convenient ELISA tools (multichannel pipettors and plate washing); no tedious separation of supernatant from pelleted resin beads, and no tubes to open and close and centrifuge
• Coated plates are 96-well strip plates, convenient for experiments requiring only a partial plate
• Easy-to-follow instructions, including detailed explanation of appropriate controls
• Three kits available, suitable for most common antibody types (mouse, rabbit, human and goat IgG subclasses) or any biotinylated antibody or “bait” protein

Applications:

• Rapid, trouble-free immunoprecipitation of multiple samples and their controls
• Immunoprecipitation when plate-based tools and techniques are preferred

How do I choose between Protein A/G and Streptavidin Coated Plate Kits?

Streptavidin is a protein that binds specifically and very strongly to biotin; therefore, the Streptavidin Coated Plate IP Kit (#45360) is appropriate for immunoprecipitation when using a biotin-labeled (biotinylated) antibody. In fact, this kit can be used to affinity purify a binding partner to any antibody species or subclass or any other protein or molecule that is biotinylated. Because the streptavidin-biotin affinity interaction is so strong, the elution step generally will dissociate only the antigen (binding partner), not the biotinylated antibody or “bait” protein.

Protein A and Protein G are different proteins that bind to immunoglobulins (primarily only IgG). Typically, Protein A is preferred for use with Rabbit polyclonal antibodies, while Protein G is preferred for use with mouse antibodies (especially monoclonals of the IgG1 subclass). Protein A/G is a recombinant of Protein A and Protein G that has the additive binding properties of both proteins. Compare Protein A, Protein G and other antibody-binding proteins.

Related Products:
Pierce Classic IP Kit
Pierce Crosslink IP Kit
Pierce Direct IP Kit
Pierce Co-IP Kit