Sypro r orange protein gel stain

A 10 μL reaction mixture was added to 96-well plate. Each biochemical reaction was consisted of 1 μL of 100 μM protein, 4 μL of 500 μM compound, 1 μL of 10× fluorescent dye of SYPRO(R) orange protein gel stain (Sigma-Aldrich, Saint Louis, MO, USA), 1 μL of 10× buffer (100 mM HEPES, 1500 mM NaCl, 50% glycerin and deionized water, pH of 7.5) and 3 μL of deionized water. Total DMSO concentration was restricted to 1% or less. The 96-well plate was filmed and centrifuged at 1000 r/min for 1 min at room temperature, and then incubated on ice for 30–60 min in the dark. The plate was submitted for detection using the Bio-Rad CFX96 Real-Time PCR system (Bio-Rad, Hercules, CA, USA). The parameter of temperature range was set as 30–80 °C with a step of 0.3 °C per minute. The excitation and emission filters of the SYPRO orange dye were set at 465 nm and 590 nm, respectively. The melting temperature (T_m) was calculated by fitting the melting curve to Boltzmann equation using GraphPad Prism 5. ΔT_m represents the difference of T_m values for the tested reactions and the blank reaction. The experiments were performed in triplicates. The expression and purification of BRD4 BD1 proteins were carried out as previously described [34 (link), 35 (link)].