Anti dykddddk flag tag antibody beads

HEK293T cells transfected with the indicated expression plasmids by the jetPRIME reagent (Polyplus) for 24 h were lysed in lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2.5 mM MgCl₂, 0.5% NP-40, 1 mM PMSF, and PIC) containing 10 mM nicotinamide and 1 μM TSA by six passes through a 29-G needle. After centrifugation at 14,000 × g for 10 min at 4 °C, the cell lysate (500 μg) was subjected to immunoprecipitation overnight at 4 °C with anti-DYKDDDDK (FLAG) tag antibody beads (clone 1E6, Wako Pure Chemical Industries), and the resins were washed five times with lysis buffer. Precipitates were examined by western blotting with the indicated antibodies.

About 1 g of N. benthamiana leaves infiltrated with pure water or Agrobacterium cells harboring pGWB511-TP-UnaG-FLAG was collected. All subsequent procedures were performed at 4°C. The leaves were ground in a mortar and pestle with 5 ml of 1× phosphate-buffered saline (PBS; pH 7.4). The homogenate was filtered through two layers of Miracloth (Merck) and centrifuged at 3000g for 10 min. The supernatant was mixed with 100 μl of anti-DYKDDDDK (FLAG) tag antibody beads (Wako) and rotated for 5 hours. The mixture was transferred to a Poly-Prep Chromatography Column (Bio-Rad) to trap the affinity beads, which were subsequently washed twice with 5 ml of 1× PBS. The affinity beads were resuspended in 150 μl of 1× PBS, and UnaG florescence was observed under blue light. Further analysis was performed with UnaG bound to the affinity beads. Immunoblot analysis was carried out as described previously using an anti-DYKDDDDK (FLAG) antibody (Wako) and an anti-mouse antibody (Thermo Fisher Scientific) (56 (link)). Fluorescence excitation and emission spectra of ipUnaG were acquired using an F-2700 fluorescence spectrophotometer (Hitachi).