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Development of a non‐isotopic saturation binding assay for analysis of dynamic interactions of flagellar export apparatus proteins
Author(s) -
Kraft Lewis,
Nguyen Viet Q.,
McMurry Jonathan L.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1006.2
Subject(s) - western blot , chemistry , flagellum , saturation (graph theory) , blot , binding protein , biophysics , microbiology and biotechnology , biochemistry , biology , gene , mathematics , combinatorics
FliI is the flagellar export ATPase in Salmonella enterica. It is negatively regulated by FliH, which also likely targets FliI to the membrane‐bound export apparatus. To understand the dynamics of flagellar export, a Type III secretion process, a saturation binding study has been undertaken to ascertain affinity constants for FliI interaction with FliH and other export protein interactions using an apparatus of our own design and construction. The apparatus utilizes an affinity resin, positive pressure and a dot‐blot style immunoassay to analyze specific binding between a His‐tagged protein and an impure binding partner, eliminating the need for radiolabeling. 96 samples may be processed simultaneously, allowing for replicates and nonspecific binding to be performed under identical conditions. The apparatus has been used to determine K D for FliH (and truncations thereof) binding to FliI and is being utilized to analyze other flagellar protein interactions. This work was supported by grants from the NIH (R15GM080701), the Research Corporation (Cottrell College Science Award) and the KSU Mentor‐Protégé Program.