The development of rugged, reliable and repeatable protein purification and enrichment is essential when performing immunoprecipitation studies. One classic approach is to couple an antibody to a solid phase resin (Protein A or Protein G) and either pack the Ab-coupled material into a short column or layer the bottom of a filter cartridge with the material for flow-through retention. Once retained, impurities and non-target species are washed through. Finally, the retained species of interest are eluted in purified form and if necessary, effectively concentrated. Our laboratory has used this kit extensively for buffer and biofluid applications with consistently outstanding results.
Classic problems that come with these methods include potential antibody-resin incompatibility (low antibody-loading), resin loss from chemical de-coupling and antibody leeching into the eluate (resulting in sample complexity and impurity). Pierce has produced a line of immunoprecipitation kits to help remedy some of these processional short-comings. The Pierce ImmunoPure Plus line can be purchased as immobilized recombinant Protein A or Protein G. They have standardized the process for distribution which reduces the variability when compared to in-lab preparations. In addition to high reproducibility, Pierce has optimized the immunoaffinity chemistry in order to improve antibody loading and decrease inadvertent antibody elution, thereby improving sample purity. The Pierce process also involves a cross-linking step that generates a more rigid spacer-arm between resin and antibody, thus better exposing the antibody and improving capture efficiency. The beads are packaged as pre-cleaned slurry and in the hands of our research group, quality has proven itself to be consistently excellent.
Pierce also has an ImmunoPure IgG Elution Buffer available. The formula is proprietary, which can be problematic for research involving mass spectrometry of the eluate. However, compared to our previous elution methods involving low pH and organic solvents the Pierce ImmunoPure IgG Elution Buffer has proven to have higher elution efficiency for practically all of our protein targets. In order to bypass the potentially incompatible solvent for MS injection, a sample clean-up step was added to our procedures (dialysis, packed pipette filtration or spin column elution). The resulting mass spectra are clean and apparently free of extraneous antibody or resin traces. The Pierce ImmunoPure Kits are now used regularly for our in-tube immunoprecipitation protocols and are thereby, highly recommended.
Also available from Pierce are Pierce ‘Seize Kits’. These kits are designed as pre-packaged spin-columns that can be coupled with the immobilized Protein A/Protein G products. The kits are designed for faster, more efficient protein purifications from complex samples without the typical problems of antibody leeching and resin loss. Pierce markets this approach as extremely favorable for producing cleaner SDS-PAGE bands, however, we use it for mass spectrometry of the eluate where it proves just as applicable. Since the ‘Seize Kits’ provide the same quality as the bare ImmunoPure immobilized Protein bead technology, it certainly has been an improvement over in-tube free-bead immunoprecipitation.
Ronald A. Miller
Research Biochemist
Merck Research Laboratories
Department of Alzheimer's Disease Research