Store vial at -20° C prior to opening. Aliquot contents and freeze at -20° C or below for extended storage. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4° C as an undiluted liquid. Dilute only prior to immediate use.

This affinity purified 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.   Although it has been reported that this antibody reacts with mouse TERT (mTERT) (see Drissi, et al. 2001), the binding to mTERT is considerably weaker and less specific than the binding to hTERT (not shown).   A SY5Y cell nuclear extract can be used as a positive control. 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. 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 horseradish peroxidase- conjugated 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.

This antiserum 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.

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