A study to assess the cross-reactivity of cellulose membrane-bound peptides with detection systems: an analysis at the amino acid level.
The growing demand for binding assays to study protein-protein interaction can be addressed by peptide array-based methods. The SPOT technique is a widespread peptide-array technology, which is able to distinguish semi-quantitatively the binding affinities of peptides to defined protein targets within one array. The quality of an assay system used for probing peptide arrays depends on the well-balanced combination of screening and read-out methods. The former address the steady-state of analyte capture, whereas the latter provide the means to detect captured analyte. In all cases, however, false-positive results can occur when challenging a peptide array with analyte or detecting captured analyte with label conjugates. Little is known about the cross-reactivity of peptides with the detection agents. Here, we describe at the amino acid level the potential of (i) 5-(and 6)-carboxytetramethylrhodamine (5(6)-TAMRA), (ii) fluoresceinisothiocyanate in form of the peptide-bound fluorescein-substituted thiourea derivative (FITC), and (iii) biotin/streptavidin-POD to cross-react with individual amino acids in a peptide sequence.
Mahrenholz
Carsten C
CC
Institut für Medizinische Immunologie, Charité-Universitätsmedizin Berlin, Berlin, Germany.
Tapia
Victor
V
Stigler
Rolf D
RD
Volkmer
Rudolf
R
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D001681:Biological Assay / Q000379:methods*; D002482:Cellulose / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding
D000596:Amino Acids; D010455:Peptides; D002482:Cellulose