title: High-Sensitivity IHC Detection of Phosphorylated p27/Kip1 in Human Tissues Using Secondary Antibody Conjugated to Polymer-HRP.
abstract: A complex composed of goat anti-rabbit secondary antibody conjugated to a polymer coated with horseradish peroxidase (HRP) molecules was used to develop rapid and highly sensitive immunostaining protocol for the detection of phosphorylated p27/Kip1 (T157) in human tissues. This polymer-HRP complex produced much better sensitivity detection compared to conventional biotin-streptavidin-HRP chemistry. Using polymer-HRP made it possible to reduce primary antibody concentration, eliminate some incubation steps such as avidin-biotin blocking and incubation with separate biotinylated secondary antibodies, and shorten the incubation time with primary antibody. Specificity of the detection was confirmed by eliminating labeling after treating tissues with lambda phosphatase to remove phosphate groups from p27/Kip1. Secondary antibodies conjugated to polymer-HRP is a reagent of choice in both research and diagnostic pathology allowing detecting low abundant and weakly expressed tissue targets.
authors.affiliation: Bio-Techne, Inc., 614 McKinley Place NE, Minneapolis, MN, 55413, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Grahek
authors.affiliation: Bio-Techne, Inc., 614 McKinley Place NE, Minneapolis, MN, 55413, USA.
authors.forename: Ana
authors.initials: A
authors.lastname: Ptak
authors.affiliation: Immunocytochemistry & Elispot Assays, Bio-Techne, Inc.,, Minneapolis, MN, USA. alex.kalyuzhny@Bio-Techne.com.
authors.forename: Alexander E
authors.initials: AE
authors.lastname: Kalyuzhny
mesh_terms: D045163:Antibodies, Phospho-Specific / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000276:immunology / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D018796:Immunoconjugates; D007150:Immunohistochemistry / Q000379:methods*; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D012680:Sensitivity and Specificity
chemical_list: D045163:Antibodies, Phospho-Specific; D018796:Immunoconjugates; D010750:Phosphoproteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D006735:Horseradish Peroxidase
keywords: Antigen retrieval; HRP; Horseradish peroxidase; Immunohistochemistry; Lambda phosphatase; Phospho-specific antibodies; Secondary antibodies conjugated to polymer-HRP; Streptavidin-HRP; TMAs; Tissue microarrays; p27/Kip1