Weak protein–protein interactions revealed by immiscible filtration assisted by surface tension

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 interaction...

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Bibliographic Details
Published in:Analytical biochemistry 2014-02, Vol.447, p.133-140
Main Authors: Berry, Scott M., Chin, Emily N., Jackson, Shawn S., Strotman, Lindsay N., Goel, Mohit, Thompson, Nancy E., Alexander, Caroline M., Miyamoto, Shigeki, Burgess, Richard R., Beebe, David J.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Antibodies, Monoclonal - metabolism
Cell Line
Exclusion-based sample preparation
Filtration - methods
Glycogen synthase kinase-3β
Green fluorescent protein
Green Fluorescent Proteins - chemistry
Green Fluorescent Proteins - isolation & purification
Green Fluorescent Proteins - metabolism
Immunoprecipitation
Low-density lipoprotein receptor
Mice
Polyol-responsive
Protein Binding
Proteins - isolation & purification
Proteins - metabolism
Surface Tension
Time Factors
Wnt Signaling Pathway
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Summary: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.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2013.10.038