title: Using Phospho-Peptides Immobilized on Magnetic Beads for Absorption Control in Immunohistochemistry.
abstract: Phospho-specific primary antibodies are used in immunohistochemistry (IHC) to detect phosphorylated sequences in proteins, in some cases they may also cross-react with non- or de-phosphorylated sequences. To rule out nonspecific staining, and to determine that the staining pattern is specific it is necessary to employ a so-called absorption control: phospho-specific primary antibodies are first incubated with phospho-peptide immunogen to block antibody binding sites, and this mixture is applied to tissue sections. If the antibody pre-blocked with cognate immunogen does not produce tissue staining, then the antibody is considered specific. However, if the staining does occur, it indicates that the antibody is nonspecific. The drawback of doing absorption by mixing the peptide with the antibody is that in solution such peptide-antibody complexes can dissociate unblocking the antibody which becomes capable of binding to cell and tissue targets, producing unwanted staining. To overcome this problem, we have developed a simple absorption control technique allowing for efficient blocking of phospho-specific antibodies with phospho-peptides immobilized on magnetic beads. This technique allows for sequestration of peptide-antibody complex from the incubation mixture eliminating the risk of un-blocking primary antibodies via their dissociation from the blocking peptide.
authors.affiliation: Cell IDx, 6197 Cornerstone Court East, Ste 102, San Diego, CA, 92121, USA.
authors.forename: David
authors.initials: D
authors.lastname: Schwartz
authors.affiliation: Bio-Techne, Inc.,, Minneapolis, MN, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Grahek
authors.affiliation: Innopep, Inc., San Diego, CA, USA.
authors.forename: Yingwei
authors.initials: Y
authors.lastname: He
authors.affiliation: Innopep, Inc., San Diego, CA, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Vertex Pharmaceuticals, San Diego, CA, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Nguyen
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: D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000276:immunology; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry / Q000379:methods*; D007163:Immunosorbent Techniques; D051379:Mice; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells; D010748:Phosphopeptides / Q000276:immunology / Q000378:metabolism*
chemical_list: D045163:Antibodies, Phospho-Specific; D010748:Phosphopeptides
keywords: 3T3 cells; Absorption control; Confocal microscopy; Fluorescence immunohistochemistry; Magnetic beads; NorthernLights™ Guard anti-fade mounting media; Phospho-peptides; Phospho-specific antibodies