How to pull down intact protein complexes
|If you are using techniques such as Sepharose® beads and spin columns for pulldown, note that your protein complexes can dissolve from:|
- Exposure to large surfaces
- Mechanical strain (e.g., centrifugation)
- Excessive handling (preclearing)
Although some researchers choose to preclear using Sepharose® beads, there can be nonspecific binding interactions that can contaminate the final product. Skip unnecessary steps and make sure you get intact protein complexes. Start using Dynabeads® for protein isolation and protein complex isolation.
Sepharose® is a trademark of GE Healthcare companies.
Protein complexes remain intact and functional
- Quick and easy pulldown of intact, functional protein complexes
- No time-consuming preparation steps
- Only isolate the proteins you want
- Can be adapted for high-throughput applications
The Dynabeads® Method
Figure 1. Isolation of protein complexes using Dynabeads®
- Add your specific antibody or interacting protein with tags to Dynabeads®, then immunoprecipitate your protein of interest.
- Once the beads are exposed to a magnet, they are efficiently drawn to the tube wall, taking only your protein complex with them (Figure 1).
- As the process is gentle, yet very quick, protein complexes remain intact and functional. The protein complexes can be resuspended in a small volume ready for downstream analysis with mass spectrometry, gels, etc.
Examples of Published Articles for Protein Complex Pull Down Using Dynabeads®
Dynabeads® M-270 Epoxy were incubated with anti-GFP polyclonal antibodies or IgG and added to the soluble cell lysate fraction. These antibody-coated Dynabeads® bound to the target proteins and when the tube was placed in a magnet, were drawn to the tube wall. After washing, the isolated protein complexes were eluted from the beads, frozen with liquid nitrogen, and left to dry overnight by vacuum centrifugation. The pellet was put in SDS-PAGE buffer, separated on a precast 1-D gel before being stained with Coomassie® blue, prior to mass spectrometry. This protocol can be applied to sample materials such as viruses, bacteria, yeast, mammalian tissue, cultured cells, and mice.
2. Devos, D. et al. (2004) Components of coated vesicles and nuclear pore complexes share a common molecular architecture. PLoS Biol 2(12): e380.
Wanted to study the nuclear pore complex (NPC), the gate that mediates traffic of macromolecules across the nuclear envelope. They used computational and biochemical means to analyze the seven proteins in one of the subcomplexes in the NPC. As part of the study, they performed proteolytic mapping of domain boundaries and loop locations in the seven yeast extract nups, the set of proteins that make up the NPC. The Protein A–tagged nups were bound to Dynabeads® M-270 Epoxy with proteolytically resistant tags and purified from yeast extracts with magnetic separation. Endoproteinases were added to hydrolyze peptide bonds and any proteolytic fragments on the beads were separated by SDS-PAGE. Cleavage sites were determined by amino-terminal Edman sequencing or by estimating the molecular weight of the fragments.
3. Khalili-Shizari, et al. (2005) PrP glycoforms are associated in a strain-specific ratio in native PrPSc. J General Virology 86: 2635–2644.were investigating prion diseases and how the normal cellular prion protein PrP is converted into an abnormal isoform (PrPSc) in prion-diseased brains. They produced monoclonal antibodies (mAbs) for immunoprecipitation (IP) of PrPSc and the glycoforms from diseased and normal brain homogenates.
Direct IP was performed using mAbs crosslinked to Dynabeads®, mouse IgG on Dynabeads® Protein A, or biotinylated antibodies with Dynabeads® M-280 Streptavidin. Indirect IP was used to capture antibody-antigen complexes with anti-PrP mAbs IgG and Dynabeads® Protein G. They demonstrated that the differentially glycosylated native PrPSc are closely associated and immunoprecipitate together.
4. Suzuki, H. et al. (2004) In vitro pull-down assay without expression constructs. BioTechniques 37 (6): 918–919. developed an in vitro pulldown assay that uses in vitro biotinylated proteins rather than tagged proteins as pulldown drivers with Dynabeads® Streptavidin. They synthesized the biotinylated proteins by in vitro transcription-translation with biotin-lysine transfer RNA. They identified the following advantages:
•• No need to prepare plasmids for the pulldown drivers
•• Expressed proteins are likely to be soluble
•• Random position of biotin-labelled lysine residues throughout the protein mean that the fused tag does not interfere with interactions
•• Biotinylated proteins are functional in many cases and maintain their native conformations