hTERT Antibody

Telomerase is a reverse transcriptase that adds telomeric repeats (TTAGGG) n to chromosomal ends, compensating for the telomere shortening that occurs with DNA replication. In normal human somatic cells, telomerase is repressed and telomeres progressively shorten, leading to limited lifespan and senescence. Reactivation of telomerase activity is associated with human cancer and cell immortalization. Approximately 85% of human cancers, including breast, prostate, stomach, bladder, colon, and liver cancer, have telomerase activity, whereas most normal somatic cells do not. The specificity of telomerase to human cancer has led to investigations of telomerase activity and expression as a tumor marker. For example, the presence of telomerase activity in human urine has been identified as a marker for human bladder carcinoma. Human telomerase consists of three major subunits: a catalytic protein subunit called hTERT (for human Telomerase Reverse Transcriptase) , a template RNA called hTR, and telomerase-associated protein (TEP-1) . TERT and hTR are minimally required to reconstitute telomerase activity in vitro. In human cells, hTR is constitutively expressed. TERT transcription is a primary mechanism for regulation of telomerase activity. Anti-Telomerase catalytic subunit antibody has been tested for use in ELISA, 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.