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Product: Affinity Purified Anti-Telomerase catalytic subunit (hTERT) (Rabbit)
Code: 600-401-252 Lot #: 45jf054
Size: 100 g Physical State: Liquid (sterile filtered)
Antibody Concentration: 1.0 mg/ml (by UV absorbance at 280 nm)
Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2
Stabilizer: None Preservative: 0.01% (w/v) Sodium Azide
Storage Conditions: Store vial at -20° C. For extended storage aliquot contents and freeze at -20° C or below. Avoid cycles of freezing and thawing. Dilute only prior to immediate use. Expiration date is one (1) year from date of opening.
Background Information: 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.
Application Note(s): This affinity purified has been tested for use in immunoblotting (figure 1), immunoprecipitation (figure 2), and immunofluorescence microscopy (figure 3). In these assays, the antibody detects ectopically-expressed hTERT and high levels of endogenous hTERT. Although it has been reported that this antibody reacts against 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.
Recommended Dilutions : In immunoblot assays, whole cell or nuclear extracts were loaded at a concentration of 100g 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 l of protein A beads and 2 l of the anti-TERT serum per 1mg protein from cell lysate. A working dilution of 1:500 is also suggested for immunohistochemistry.
Purity and Specificity: This antiserum primarily detects hTERT, but several non-specific bands appear on immunoblots (Figure 1). In immunofluorescence microscopy assays, staining with anti-TERT-16 was specific to nuclei of cells with ectopic TERT expression.
Immunogen: This affinity purified antibody was prepared from whole rabbit serum produced by repeated immunizations with a synthetic peptide corresponding to amino acids 1104-1123 of hTERT (genome data base accession number AF018167). Peptide sequence: S-R-K-L-P-G-T-T-L-T-A-L-E-A-A-A-N-P-A-L
Drissi, R., Zindy, F., Roussel, M. F., and Cleveland, J.L. (2001) c-MYC-mediated regulation of telomerase activity is disabled in immortalized cells. J Biol Chem 276:32, 29994-30001.
Shay JW, Zou Y, Hiyama E, Wright WE. (2001) Telomerase and cancer. Hum Mol Genet 2001; 10:677-85.
White LK, Wright WE, Shay JW. (2001) Telomerase inhibitors. Trends Biotechnol 2001; 19:114-20.
Tollefsbol T.O., Andrews, L.G. (2001) Mechanisms for telomerase gene control in aging cells and tumorigenesis. Med Hypotheses 56: 630-7.
Hiyama E, Hiyama K, Yokoyama T, Shay JW. (2001) Immunohistochemical detection of telomerase (hTERT) protein in human cancer tissues and a subset of cells in normal tissues. Neoplasia 2001; 3:17-26.
Wick M, Zubov D, Hagen G. (1999) Genomic organization and promoter characterization of the gene encoding the human telomerase reverse transcriptase (hTERT). Gene 1999; 232:97-106.
Kolquist KA, Ellisen LW, Counter CM, Meyerson M, Tan LK, Weinberg RA, Haber DA, Gerald WL. (1998) Expression of TERT in early premalignant lesions and a subset of cells in normal tissues. Nat Genet 19:182-6.
Complete Genomic Sequence The Human Telomerase Gene
Telomere Protein Database
Human telomerase components, telomere proteins, and proteins involved in the repair of telomeric DNA
Note: This product is for research use only and is not intended for therapeutic or diagnostic applications. Please contact a technical service representative for more information.
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