A total of 293 T cells were transfected with His-ubiquitin and GFP-Aurora B or Flag-Survivin and ubiquitinated Aurora B or Survivin was immunoprecipitated from cell lysates with GFP-Trap agarose beads (Chromotek) or anti-Flag agarose beads (Sigma-Aldrich), respectively. Proteins bound on bead...
A total of 293 T cells were transfected with His-ubiquitin and GFP-Aurora B or Flag-Survivin and ubiquitinated Aurora B or Survivin was immunoprecipitated from cell lysates with GFP-Trap agarose beads (Chromotek) or anti-Flag agarose beads (Sigma-Aldrich), respectively. Proteins bound on bead...
PyTRAP-immunized mice were then challenged withPysporozoites via mosquito bite to determine if these antibodies could function in vivo to reduce liver infection. We utilized a PyGFPluc parasite, which expresses luciferase, enabling the measurement of liver-stage parasite burden by bioluminescence imaging...
The RNA–protein complexes were recovered by incubation with Ni2+ NTA Magnetic Agarose Beads (Qiagen) for 30 min. The bound sequences were isolated by total RNA extraction by using TRIzol Reagent (Life Technologies), reverse transcribed with M-MuLV Reverse Transcriptase (Roche, Indianapolis, IN...
Cells were pelleted, and the pellet was enrobed in 4% low melting point agarose, which was cut into small 1mm3 cubes. Samples were washed 3 x 15 min each with 0.1M sodium cacodylate buffer pH 7.4. Samples were fixed with freshly prepared 1.25% potassium ferrocyanide and 1% osmium ...
(R&D Systems, ARY006), protein A/G PLUS-agarose beads (Santa Cruz Biotechnology, sc-2003), GST resin (GE Healthcare, #17-0756-01), FLAG M2 affinity gel (Sigma, A2220), Strep resin (IBA, #2-1201-025), Ni-NTA agarose beads (Qiagen, #30230), GFP-Trap agarose (ChromoTek, gta-...
The lysates were sonicated on ice and clarified by centrifugation followed by preclearing with protein G agarose. The lysates were subsequently immunoprecipitated with the either anti-TDRD3 or anti-MCL-1 antibodies. Immunoprecipitated proteins were analyzed by western blots using indicated antibodies. ...
The ability of proteins to bind AMP, SZMP, or AICAR was also assayed using either a commercial AMP-agarose resin (catalog no. A1271; Merck) or SZMP- and AICAR-resins prepared as described for the ZMP resin (10). In each case, elution of interacting proteins was performed with 5 mm ...
monovalent IL-2WTFc/Fc-His pairing with a HisTrap column (GE) using an imidazole gradient elution (AKTA protein purification system, GE). Assessment of binding and activity on CTLL-2 cells. The murine CTLL-2 cell line (IL-2-dependent, CD25high) was cultured in complete RPMI 1640 (10% ...
as indicated by the reduced number of TRAP+multinucleated cells (Fig.5a, b). We performed a pit resorption assay in an osteoblast–osteoclast co-culture system to determine the osteoclast activity. Consistent with the TRAP staining results, osteoclasts in the co-culture with Klotho-deficient osteo...