hTERT (C-Term) antibody

Catalog No. ABIN6655122

Product Details

Target: hTERT

Binding Specificity: C-Term

Reactivity: Human

Host: Rabbit

Clonality: Polyclonal

Conjugate: Un-conjugated

Application: ELISA, Western Blotting (WB), Immunoprecipitation (IP), Fluorescence Microscopy (FM), Immunohistochemistry (IHC)

Purification: Affinity Purified Anti-hTERT Antibody was prepared from monospecific antiserum by immunoaffinity chromatography using synthetic peptide coupled to agarose beads. Assay by immunoelectrophoresis resulted in a single precipitin arc against anti-Rabbit Serum. 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).

Immunogen:

Immunogen: This affinity purified antibody was prepared from whole rabbit serum produced by repeated immunizations with a synthetic peptide corresponding to a region near the carboxy terminal end of hTERT (accession number AF018167).

Immunogen Type: Peptide

Isotype: IgG

Application Details

Application Notes:

Immunohistochemistry Dilution: 1:500

Application Note: 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 1 mg 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 4 °C. 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.

Immunoprecipitation Dilution: IP 2μL per mg lysate

ELISA Dilution: 1:10,000 - 1:50,000

Western Blot Dilution: 1:500

IF Microscopy Dilution: 1:500

Restrictions: For Research Use only

Handling

Format: Liquid

Buffer:

Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2

0.01 % (w/v) Sodium Azide

Stabilizer: None

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: -20 °C

Storage Comment: Store vial at -20° C or below prior to opening. This vial contains a relatively low volume of reagent (25 μL). To minimize loss of volume dilute 1:10 by adding 225 μL of the buffer stated above directly to the vial. Recap, mix thoroughly and briefly centrifuge to collect the volume at the bottom of the vial. Use this intermediate dilution when calculating final dilutions as recommended below. Store the vial at -20°C or below after dilution. Avoid cycles of freezing and thawing.

References

Bernal, Zafon, Domínguez, Bertran, Tusell: "Generation of Immortalised But Unstable Cells after hTERT Introduction in Telomere-Compromised and p53-Deficient vHMECs." in: International journal of molecular sciences, Vol. 19, Issue 7, (2018) (PubMed).

Eitan, Braverman, Tichon, Gitler, Hutchison, Mattson, Priel: "Excitotoxic and Radiation Stress Increase TERT Levels in the Mitochondria and Cytosol of Cerebellar Purkinje Neurons." in: Cerebellum (London, England), Vol. 15, Issue 4, pp. 509-17, (2017) (PubMed).

Radan, Hughes, Teichroeb, Vieira Zamora, Jewer, Postovit, Betts: "Microenvironmental regulation of telomerase isoforms in human embryonic stem cells." in: Stem cells and development, Vol. 23, Issue 17, pp. 2046-66, (2014) (PubMed).

Theurillat, Udeshi, Errington, Svinkina, Baca, Pop, Wild, Blattner, Groner, Rubin, Moch, Privé, Carr, Garraway: "Prostate cancer. Ubiquitylome analysis identifies dysregulation of effector substrates in SPOP-mutant prostate cancer." in: Science (New York, N.Y.), Vol. 346, Issue 6205, pp. 85-9, (2014) (PubMed).

Wu, Dudognon, Nguyen, Hillion, Pendino, Tarkanyi, Aradi, Lanotte, Tong, Chen, Ségal-Bendirdjian: "Immunodetection of human telomerase reverse-transcriptase (hTERT) re-appraised: nucleolin and telomerase cross paths." in: Journal of cell science, Vol. 119, Issue Pt 13, pp. 2797-806, (2006) (PubMed).

Target Details

Target: hTERT

Background:

Synonyms: rabbit anti-TERT antibody, rabbit anti-Telomerase catalytic subunit antibody, hTERT, Telomerase reverse transcriptase, HEST2, Telomerase-associated protein 2, TP2, EST2, TCS1, TRT

Background: 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.

Gene Name: TERT

Gene ID: 7015

UniProt: O14746