Heterogeneous transition metal-based fluorescence polarization (HTFP) assay for probing protein interactions.
Analyses of protein interactions are fundamental for the investigation of molecular mechanisms responsible for cellular processes and diseases, as well as for drug discovery in the pharmaceutical industry. The present study details the development of a fluorescence polarization assay using melanoma inhibitory activity (MIA) protein-binding compounds and studies of the binding properties of this protein. Since they are dependent on the the lifetime of the fluorescent label, currently available fluorescence polarization assays can only determine interactions with either high- or low-molecular weight interaction partners. Our new approach eliminates this limitation by immobilizing a known binding partner of MIA protein to a well plate and by labeling the target protein using luminescent transition metal labels such as Ru(bpy)3 for binding studies with both high- and low-molecular weight interaction partners. Due to the use of a functionalized surface, we termed our concept heterogeneous transition metal-based fluorescence polarization (HTFP) assay. The assay's independence from the molecular weight of potential binding partners should make the technique amenable to investigations on subjects as diverse as multimerization, interactions with pharmacophores, or binding affinity determination.
Institute for Organic Chemistry, University of Regensburg, Regensburg, Germany.
Alexander
A
Riechers
Jennifer
J
Schmidt
Burkhard
B
König
Anja Katrin
AK
Bosserhoff
D001639:Bicarbonates / Q000737:chemistry; D001681:Biological Assay; D001710:Biotin / Q000737:chemistry; D002021:Buffers; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D020099:Coated Materials, Biocompatible / Q000737:chemistry / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000378:metabolism; D017879:Isothiocyanates / Q000378:metabolism; D007700:Kinetics; D008545:Melanoma / Q000473:pathology; D008954:Models, Biological; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000378:metabolism*; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D009942:Organometallic Compounds / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D013696:Temperature; D013997:Time Factors; D028561:Transition Elements / Q000378:metabolism*
C546778:AR54 peptide; D001639:Bicarbonates; D002021:Buffers; D020099:Coated Materials, Biocompatible; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D017879:Isothiocyanates; C090059:MIA protein, human; D009363:Neoplasm Proteins; D009942:Organometallic Compounds; D010455:Peptides; D028561:Transition Elements; C415768:tris(2,2'-bipyridyl)ruthenium(II); D009580:Nitroblue Tetrazolium; D001710:Biotin