Anti 6 his antibody

Primary hippocampal neurons were cultured as described previously (Deng et al., 2013 (link)). The purity of neurons was determined by immunocytochemistry for βIII-tubulin, which indicated that 95% of cells in the cultures were βIII-tubulin (1:250; Millipore, Temecula, CA, USA) positive (data not shown). Then, the transduction ability of TAT-Ngn2 was assayed by an immunofluorescence analysis (Wang et al., 2009 (link)). In brief, the neurons were cultured with TAT-Ngn2 (125 μg/l) for 24 h, washed three times with PBS after fixation in 4% PFA (Dietz et al., 2002 (link)). Then incubated simultaneously with primary antibodies: anti-6× His antibody (1:2,000; Abcam, Cambridge, MA, USA) and βIII-tubulin (1:250; Millipore, Temecula, CA, USA) overnight at 4°C. The neurons were washed three times with PBS and then incubated with two secondary antibodies: FITC-labeled goat anti-rabbit IgG (1:200; Vector, Burlingame, CA, USA) and Alexa 594-labeled donkey anti-mouse IgG (1:500; Molecular Probes, Rockford, IL, USA) for 1 h at 37°C. Nuclei were visualized by DAPI staining. Images were viewed with a fluorescence microscope (BX60; Olympus). These measurements were made on cells present in six randomly selected fields in each experiment and repeated in at least three independent cultures.

For protein analyses cells were subjected to 12% (w/v) sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) as described by Laemmli (1970) (link). To visualize and confirm protein separation, 2,2,2-trichloroethanol was incorporated into the polyacrylamide gels (Ladner et al., 2004 (link)) and detected within a Gel Doc^TM EZ gel documentation system (Bio-Rad). Afterward the proteins were transferred onto nitrocellulose membranes (Whatman) which were then subjected to immunoblotting. In a first step the membranes were incubated either with 0.1 μg/mL Anti-6×His^® antibody (Abcam, Inc.) to detect EF-P, or with 0.25 μg/ml Anti-Arg^Rha antibody (Li et al., 2016 (link); Krafczyk et al., 2017 (link)) to visualize rhamnosylation. These primary antibodies (rabbit) were then targeted with 0.2 μg/ml Anti-rabbit alkaline phosphatase-conjugated secondary antibody (Rockland). Localization was visualized by adding development solution [50 mM sodium carbonate buffer, pH 9.5, 0.01% (w/v) p-nitro blue tetrazolium chloride (NBT) and 0.045% (w/v) 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)].