Biological mechanisms are often mediated by transient interactions between multiple proteins. The isolation of intact protein complexes is essential to understanding biochemical processes and an important prerequisite for identifying new drug targets and biomarkers. However, low-affinity interactions are often difficult to detect. Here, we use a newly described method called immiscible filtration assisted by surface tension (IFAST) to isolate proteins under defined binding conditions. This method, which gives a near-instantaneous isolation, enables significantly higher recovery of transient complexes compared to current wash-based protocols, which require reequilibration at each of several wash steps, resulting in protein loss. The method moves proteins, or protein complexes, captured on a solid phase through one or more immiscible-phase barriers that efficiently exclude the passage of nonspecific material in a single operation. We use a previously described polyol-responsive monoclonal antibody to investigate the potential of this new method to study protein binding. In addition, difficult-to-isolate complexes involving the biologically and clinically important Wnt signaling pathway were isolated. We anticipate that this simple, rapid method to isolate intact, transient complexes will enable the discoveries of new signaling pathways, biomarkers, and drug targets.
|Number of pages||8|
|State||Published - Feb 15 2014|
Bibliographical noteFunding Information:
This work was funded by the UW Stem Cell and Regenerative Medicine Center, the Walter H. Coulter Translational Research Partnership, NIH Grants 5R33CA137673 (D.B.) and 5R01CA077474 and 5R01GM083681 (S.M.), and the Bill & Melinda Gates Foundation through the Grand Challenges in Global Health initiative. M. Goel is a Khorana Scholar. We thank Anna Lazic (Nanotemper Technologies) for help with the microscale thermophoresis. We also thank Catharine Moran for fabricating IFAST devices and Tye Gribb for donating IFAST devices. D.J.B. holds equity in Bellbrook Labs, LLC, Ratio, Inc., and Salus Discovery LLC. S.M.B., L.N.S., and R.R.B. hold equity in Salus Discovery LLC. R.R.B. and N.E.T. hold equity in NeoClone, LLC, which markets mAb 8RB13.
- Exclusion-based sample preparation
- Glycogen synthase kinase-3β
- Green fluorescent protein
- Low-density lipoprotein receptor
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology