3 articles published in JoVE
Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery Misbha Ud Din Ahmad1, Alexander Fish1, Jeroen Molenaar1, Sridhar Sreeramulu2, Christian Richter2, Nadide Altincekic2, Harald Schwalbe2, Hans Wienk1, Anastassis Perrakis1 1Oncode Institute and Division of Biochemistry, the Netherlands Cancer Institute, 2Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.
A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice Else A. Aalbersberg1, Martine M. Geluk-Jonker1, Thirumaraichelvi Young-Mylvaganan1, Linda J. de Wit-van der Veen1, Marcel P. M. Stokkel1 1Department of Nuclear Medicine, Netherlands Cancer Institute Well-differentiated neuroendocrine tumors overexpress somatostatin receptors which can be utilized for diagnostic imaging with the radiolabeled somatostatin analog 68Ga-DOTATATE. This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiation safety and time constrictions due to the short half-life of 68Ga are taken into account.
Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons David Svilar1,2, Conchita Vens3, Robert W. Sobol1,2,4 1Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, 2Hillman Cancer Center, University of Pittsburgh Cancer Institute, 3Department of Experimental Therapy, The Netherlands Cancer Institute, 4Department of Human Genetics, University of Pittsburgh School of Public Health We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.