Mouse anti his tag antibody

The cytotoxicity of E1h was determined by 3-(3,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay as described [40 (link)]. Cells (10,000 per well) cultured for 1 day in 46-well plates were replaced with medium containing 100 nM E1h after washed with PBS, and further cultured. The indicated time point later, the surviving cells were stained with MTT and quantified by absorbance at 540 nm. The MTT assay results were plotted with mean ± S.D. of three experiments.
The in vivo stability of E1h was measured by western blotting against E1h in tumor tissue obtained from PC3-bearing mice after in vivo optical imaging. The tumor tissues dissected from mice were soaked in PBS buffer containing 1X protease inhibitor cocktail solution (GenDEPOT) and grinded with homogenizer. The tumor samples were centrifuged in 12,000 rpm for 10 min. The supernatants (100 μg protein) were mixed with SDS sample buffer and separated with 12% SDS-PAGE. After transferred to nitrocellulose membranes, proteins were first probed using a mouse anti-his tag antibody (1:1000 dilution, Santa Cruz Biotechnology, CA) and then a horseradish peroxidase-conjugated anti rabbit secondary antibody (1:2000 dilution, Amersham, UK). As a loading control, beta-actin was detected using the same (stripped) membrane.

SDS-PAGE and Western blotting were performed as described with some modifications (Ausubel 2003 ). The protein expression was confirmed by analysing in 12.5% SDS-PAGE gel, followed by Western blotting. The proteins from the gel were transferred to the nitrocellulose membrane before probing with mouse-anti-His-tag antibody (Santa Cruz) and finally, detecting by rabbit-anti-mouse IgG-HRP (Santa Cruz).

Intracellularly delivered proteins were visualized by immunocytochemical staining for His-Tag, as described previously^{12 (link),13 (link)}. Briefly, cells were grown on coverslips and incubated with 5.0 μM Tat-CHIP and Con-CHIP for 1 h. Thereafter, cells were fixed with 4% paraformaldehyde for 5 min at 25 °C, and immunocytochemical staining was conducted using mouse anti-His-Tag antibody (1:200, Santa Cruz Biotechnology) and Alexa Fluor 488-conjugated anti-mouse IgG (1:1,000, Invitrogen, Carlsbad, CA). Cells were counterstained with 1 μg/mL 4,6-diamidino-2-phenylindole (Roche Applied Science, Mannheim, Germany).