Alphascreen igg protein a detection kit

The AlphaScreen assays with the recombinant proteins described above were performed as described previously40 (link), with several modifications. The AlphaScreen assays were performed using a total volume of 15 μL containing 100 mM Tris-HCl (pH8.0), 100 mM NaCl, 0.1% Tween20, 1 mg/mL BSA, 1 μL biotinylated proteins, and FLAG-tagged proteins at 25 °C for 1 h in a 384-well Optiplate (PerkinElmer). Using the AlphaScreen IgG (ProteinA) detection kit (PerkinElmer) instruction manual, 10 μL of the detection mixture containing 100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.1% Tween20, 1 mg/mL BSA, 5 μg/mL anti-DYKDDDDK antibody (1E6, Wako Pure Chemical Industries, Ltd. Osaka, Japan), 0.1 μL streptavidin-coated donor beads, and 0.1 μL Protein A-coated acceptor beads were added to each well of the 384-well Optiplate, followed by incubation at 25 °C for 1 h. Luminescence was analysed using the AlphaScreen detection program.

In vitro cleavage activity assays of HIV-1 PR were carried out in a total volume of 15 μl consisting of 100 mM Tris–HCl pH 8.0, 0.01% Tween-20, 1 mg/ml BSA, 1 μl crude recombinant protease (∼ 0.75 μM) and 0.5 μl crude recombinant FLAG-biotin-tagged CA/NC (∼ 0.037 μM) at 37°C for 1 h in a 384-well Optiplate (PerkinElmer, Boston, MA, United States). To assay the effects of HIV-1 PR on various human protein kinases, 3 μl HIV-1 PR and human PK each was incubated at 37°C for 10 min, FLAG-biotin-tagged CA/NC or GST-biotin-tagged p2–p7 was added and the reaction further incubated at 37°C for 1 h in a 384-well Optiplate. In accordance with the AlphaScreen IgG (Protein A) detection kit (PerkinElmer) instruction manual, 10 μl of detection mixture containing 100 mM Tris–HCl pH 8.0, 0.01% Tween-20, 1 mg/ml BSA, 5 μg/ml Anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO, United States) or Anti-GST antibody (GE Healthcare, Buckinghamshire, United Kingdom), 0.1 μl streptavidin-coated donor beads and 0.1 μl anti-IgG (Protein A) acceptor beads were added to each well followed by incubation at 26°C for 1 h. Luminescence was analyzed by the AlphaScreen detection program. Each assay was performed in triplicate, and the data represent the means and standard deviations of three independent experiments.

AlphaScreen assays were performed as described previously [23 (link)]. All recombinant proteins used here was synthesized using a wheat germ based cell-free system as described above. For each protein kinase, 1 μl of crude recombinant biotinylated construct from the human kinase library was incubated with 1 μl of crude GST-Gag or GST-DHFR in 10 μl of kinase assay buffer (100 mM Tris–HCl pH8.0, 10 mM MgCl₂, 0.1% Tween20, 0.1% BSA) at 37°C for 1 h in one well of a 384-well Optiplate (Perkin Elmer, Foster City, CA. In accordance with the AlphaScreen IgG (protein A) detection kit (Perkin Elmer) instruction manual, 15 μl of detection mixture containing 100 mM Tris–HCl pH 8.0, 0.01% Tween-20, 1 mg/ml BSA, 5 μg/ml Anti-FLAG antibody (GE healthcare, Buckinghamshire, UK), 5 ng streptavidin-coated donor beads and 5 ng anti-IgG (protein A) acceptor beads were added to each well followed by incubation at 26°C for 1 h. AlphaScreen signals from the mixture were detected using an EnVision device (PerkinElmer) with the AlphaScreen signal detection program.