HIV-1 integrase (IN) is an important therapeutic target as its function is essential for the viral lifecycle. methods include a homogenous time-resolved fluorescence-based assay which allows for measuring EC50 values for the inhibitor-induced aberrant IN multimerization a dynamic light scattering-based assay which allows for monitoring the formation and sizes of oligomeric IN particles in a time-dependent manner and a chemical cross-linking-based assay of interacting IN subunits which Exemestane allows for the determination of IN oligomers in viral particles. is the compound concentration is the HTRF signal is the inhibitor IC50 and is the Mouse monoclonal antibody to SAFB1. This gene encodes a DNA-binding protein which has high specificity for scaffold or matrixattachment region DNA elements (S/MAR DNA). This protein is thought to be involved inattaching the base of chromatin loops to the nuclear matrix but there is conflicting evidence as towhether this protein is a component of chromatin or a nuclear matrix protein. Scaffoldattachment factors are a specific subset of nuclear matrix proteins (NMP) that specifically bind toS/MAR. The encoded protein is thought to serve as a molecular base to assemble a′transcriptosome complex′ in the vicinity of actively transcribed genes. It is involved in theregulation of heat shock protein 27 transcription, can act as an estrogen receptor co-repressorand is a candidate for breast tumorigenesis. This gene is arranged head-to-head with a similargene whose product has the same functions. Multiple transcript variants encoding differentisoforms have been found for this gene. Hill slope. Fig. 3 Example data set for HTRF-based IN multimerization assay. HTRF data obtained with increasing concentration of BI-1001 ) 8 min () and 30 min () after addition of MINI KF116. Recorded signals indicate an equilibrium shift toward higher order oligomers … 3.3 IN Multimerization in Viral Particles 3.3 Generation Isolation and Lysis of Viral Particles Seed 2 × Exemestane 106 HEK293 cells in 10 ml complete medium in a 100 mm tissue-culture dish and culture overnight at 37 °C and 5 % CO2. Next day transfect cells with HIV-1 proviral plasmid (for 5 min at room temperature to pellet the cell debris. Collect the cell-free virus-containing supernatant and filter it through 0.45 μm sterile filter. Aliquot 25 μl of virus-containing filtered supernatant in an Eppendorf tube and store the rest at 4 °C. Use 25 μl of virus-containing filtered supernatant to perform HIV-1 p24 ELISA using the manufacturer’s kit and protocol. Generate the standard curve in the range of 7.8–125 pg/ml of HIV-1 p24 using HIV-1 p24 antigen supplied with the kit. Calculate the volume of virus-containing filtered supernatant equivalent to 1000–1500 ng of HIV-1 p24 using the HIV-1 p24 standard curve. Aliquot the calculated volume of virus-containing filtered supernatant in a new 15 ml tube and bring the volume up to 12 ml with complete medium. Load 12 ml of virus-containing filtered supernatant in a 13.2 ml ultracentrifuge tube. Gently underlay 1 ml of 25 % sucrose solution using a Pasteur pipette. Load the ultracentrifuge tube in the swinging bucket rotor. Ultracentrifuge at 135 0 × for 2 h at 4 °C. Decant the supernatant and carefully wipe Exemestane the inside of the tube with rolled-up Kimwipes to remove traces of supernatant and sucrose. Avoid touching the bottom of the tube. Add virion lysis buffer to adjust the concentration of virions to 15 ng/μl of HIV-1 p24. For example if supernatant equivalent to 1500 ng of HIV-1 p24 was pelleted then add 150 μl of virion lysis buffer. Incubate the tube at 37 °C for 15 min briefly vortex the tube to dislodge the viral pellet and Exemestane resus-pend by pipetting. Collect the lysed virions in a new Eppendorf tube. 3.3 Virion-Associated IN Cross-Linking Reaction In an Eppendorf tube add lysed virions equivalent to 50 ng of HIV-1 p24 and the calculated volume of conjugation buffer. Prepare 200 μM BS3 cross-linking solution (as previously described [22]. The concentration of the purified proteins must be maintained between 10 and 30 μM in the storage buffer (50 mM HEPES pH 7.5 1 M NaCl 7.5 mM CHAPS 2 mM β-mercaptoethanol and 10 % glycerol) to avoid auto-aggregation. Purified recombinant INs are aliquoted into small volumes flash-frozen by liquid N2 immersion and stored at ?80 °C. Importantly once thawed the protein aliquot must be used immediately or discarded. 2 BSA must be of TRF grade (Perkin Elmer.