The use of SPR to mea sure binding parameters for interactions is widely reported. Many applications range from purification, epitope mapping, and ligand fishing to identifying small molecules in a screening mode achieved Tubacin chemical structure by measuring reaction kinetics, and binding constants. Directly monitoring the binding of low molecular mass compounds to immobilized macromolecules has had sig nificant impacts on pharmaceutical discoveries. Methods were developed for TopI DNA cleavable com plex detection to verify TopI inhibitor activity. SPR was recently used in TopI inhibition studies. How ever, most of those immobilized small molecules or short sequence nucleotides were used as ligands on sen sor chips, and TopI was used as the analyte that flowed through the sensor chip.

TopI protein preparation Inhibitors,Modulators,Libraries is much more complicated than that for DNA, and large quantities of analytes are Inhibitors,Modulators,Libraries consumed with large scale screening using SPR. It would be beneficial to develop an SPR assay with TopI immobilized onto the sensor chip as the ligand to detect TopI DNA cleavage complexes in response to a variety of analytes. Methods Reagents and antibodies Camptothecin and evodiamine were pur chased from Sigma Aldrich. Enhanced chemiluminescence reagents were pur chased from PerkinElmer. A Plas mid Midiprep Kit was obtained from Promega. All solvents used in this study were from Merck or Sigma Aldrich. Recombinant human TopI protein expression and purification Complementary DNAs encoding Inhibitors,Modulators,Libraries full length hTop I were subcloned into the baculoviral expression vectors, pFastBac HTa and pFastBac HTc.

The bacmid constructs were prepared using a Bac to Bac baculovirus expression system protocol. To express and purify the recombinant hTopI, a recombinant baculoviral stock was used to infect 2 107 Sf 9 insect cells per 140 mm plate. Infected cells were Inhibitors,Modulators,Libraries cultured at 27 C for 3 days. An Ni NTA column/imidazole was used for hTopI fractionation. Western blot analysis Purified protein samples were resolved by sodium dode cylsulfate polyacrylamide gel electrophoresis and electrotransferred onto a polyvinylidene dif luoride membrane. The membrane was incubated with a primary rabbit antibody against hTopI or H2AX, respectively, at 4 C overnight, and then incubated with a horseradish peroxidase conjugated secondary immunoglobulin G antibody. the immunoreactive Inhibitors,Modulators,Libraries bands were visualized with PerkinElmer ECL reagents.

Comet assay The comet assay is a widely used sellectchem method to analyze the consequence of TopI inhibition of DNA integrity, since it enables DNA strand breaks to be detected with high sen sitivity at the single cell level. TopI cleavage complexes are characterized by TopI concealed single strand breaks. When TopI is digested by proteasomes, the sin gle strand breaks collide with replication runoff to form DNA double strand breaks on the leading strand.