title: Detection and quantification of apoptosis in primary cells using Taqman® protein assay.
abstract: There are several methods to detect apoptosis using cleaved caspase-3 and each harbors its own advantages and disadvantages. When primary cell cultures are used, the disadvantages of the standard methods can make apoptosis detection difficult due to their slow growth rate and replicative senescence, thereby limiting the available cell number and experiment time span. In this chapter, we describe apoptosis detection and quantification using an innovative method named TaqMan(®) protein assay. TaqMan(®) protein assay uses antibodies and proximity ligation for quantitative real-time PCR. Biotinylated antibodies are labeled with oligonucleotides. When the labeled antibodies bind in close proximity, the oligonucleotides are connected using DNA ligase. The ligation product is amplified and detected using Taqman(®) based Real-Time PCR. Using this technique, we can not only detect apoptosis with a 1,000-fold higher sensitivity than western blot, but we can also exactly quantify cleaved caspase-3 expression. Thereby apoptosis can be determined and quantified in a fast reliable manner.
authors.affiliation: Department of Neurosurgery, University of Tuebingen, Hoppe-Seyler-Str.3, 72076, Tuebingen, Germany, christina.pfister@med.uni-tuebingen.de.
authors.forename: Christina
authors.initials: C
authors.lastname: Pfister
authors.forename: Heike
authors.initials: H
authors.lastname: Pfrommer
authors.forename: Marcos S
authors.initials: MS
authors.lastname: Tatagiba
authors.forename: Florian
authors.initials: F
authors.lastname: Roser
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D017209:Apoptosis; D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D019921:Biotinylation; D017364:Collagenases / Q000737:chemistry; D006801:Humans; D010447:Peptide Hydrolases / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012984:Software; D046509:Tissue Culture Techniques
chemical_list: D000906:Antibodies; D011506:Proteins; C575294:accutase; D010447:Peptide Hydrolases; D017364:Collagenases