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 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.
Department of Biomedical Engineering, University of Wisconsin at Madison, Madison, WI 53705, USA. Electronic address: scott.michael.berry@gmail.com.
Scott M
SM
Berry
Department of Oncology, University of Wisconsin at Madison, Madison, WI 53705, USA.
Emily N
EN
Chin
Department of Oncology, University of Wisconsin at Madison, Madison, WI 53705, USA.
Shawn S
SS
Jackson
Department of Biomedical Engineering, University of Wisconsin at Madison, Madison, WI 53705, USA.
Lindsay N
LN
Strotman
Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India.
Mohit
M
Goel
Department of Oncology, University of Wisconsin at Madison, Madison, WI 53705, USA.
Nancy E
NE
Thompson
Department of Oncology, University of Wisconsin at Madison, Madison, WI 53705, USA.
Caroline M
CM
Alexander
Department of Oncology, University of Wisconsin at Madison, Madison, WI 53705, USA.
Shigeki
S
Miyamoto
Department of Oncology, University of Wisconsin at Madison, Madison, WI 53705, USA.
Richard R
RR
Burgess
Department of Biomedical Engineering, University of Wisconsin at Madison, Madison, WI 53705, USA.
David J
DJ
Beebe
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002460:Cell Line; D005374:Filtration / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D013500:Surface Tension; D013997:Time Factors; D060449:Wnt Signaling Pathway
D000911:Antibodies, Monoclonal; D011506:Proteins; D049452:Green Fluorescent Proteins
Exclusion-based sample preparation; Glycogen synthase kinase-3β; Green fluorescent protein; Immunoprecipitation; Low-density lipoprotein receptor; Polyol-responsive