Anti-Telomerase catalytic subunit (RABBIT) Antibody, 100 g, Liquid (sterile filtered)

Anti-Telomerase catalytic subunit antibody has been tested for use in immunoblotting, immunoprecipitation, and immunofluorescence microscopy. In these assays, the antibody detects ectopically-expressed hTERT and high levels of endogenous hTERT. A SY5Y cell nuclear extract can be used as a positive control. This antibody primarily detects hTERT, but several non-specific bands appear on immunoblots. In immunofluorescence microscopy assays, staining with anti-TERT-16 was specific to the nuclei of cells with ectopic TERT expression. In immunoblot assays, whole cell or nuclear extracts were loaded at a concentration of 100 μg protein per well. A working dilution of 1:500 anti-TERT antibody was used followed by a 1:3,000 dilution of HRP goat anti-rabbit IgG as the secondary antibody. For immunofluorescence microscopy staining, a working dilution of 1:500 was used followed by a 1:200 dilution of rhodamine-conjugated donkey anti-rabbit IgG as a secondary antibody. Immunoprecipitation was performed using 20 μL of protein A beads and 2 μL of the anti-TERT serum per 1mg protein from cell lysate. A working dilution of 1:500 is also suggested for immunohistochemistry. To detect TERT, fix cells in 2% paraformaldehyde (in PBS) for 10'. Wash the slides twice in PBS for 5' each. Permeabilize the cells in 0.5% NP-40 for 10'. Wash as before in PBS. Block the cells using PBG buffer (0.2% cold water fish gelatin (Sigma G-7765) and 0.5% BSA in PBS) for 20' at room temperature. Incubate in primary antibody (diluted in PBG) for 1-2 hours at RT or overnight at 4oC. Wash the slides three times in PBG for 5' each. Incubate with secondary antibody (diluted in PBG) for 1 hour at RT in the dark. Wash the slides three times in PBG for 5' each. Mount in DAPI-containing medium.

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