TeloTAGGG™ Telomerase PCR ELISA 11854666910

Photometric enzyme immunoassay for the detection of telomerase activity, utilizing the Telomeric Repeat Amplification Protocol (TRAP).
Telomeres are specialized DNA protein structures found at the end of eukaryotic chromosomes. Telomeric DNA is characterized by an array of tandemly repeated, G-rich DNA sequences (six nucleotides in length) that are highly conserved during evolution (human repeat sequence: TTAGGG). As DNA polymerase a is unable to replicate the very ends of linear DNA, every replication cycle leads to a progressive shortening of the telomeric ends in normal somatic cells. This phenomenon appears to be linked to the limited proliferative capacity of normal somatic cells of higher eukaryotes. Telomerase, a ribonucleoprotein, catalyzes the addition of TTAGGG repeats to the ends of vertebrate chromosomes, using a complementary sequence of its intrinsic RNA component as a template. Telomerase activity has been shown to be expressed in germ cells, as well as most cell lines and tumors. Escaping from the proliferative limitations of cellular senescence, telomerase reactivation may be a prerequisite of the development of malignant tumor cells from somatic cells.
The TeloTAGGG™ Telomerase PCR ELISA is designed for use in life science research studies for the highly sensitive qualitative detection of telomerase activity in cell extracts from cell cultures and other biological samples. The TeloTAGGG Telomerase PCR ELISA utilizes a single-tube reaction mixture that simplifies the amplification reaction. The ELISA technique uses a biotinylated primer to immobilize the TRAP reaction products within a streptavidin-coated microplate, and a specific DIG-labeled probe for detection.
Features and Benefits

• Easy: One-step/one-tube ready-to-use reaction mix for elongation and amplification. Detection of up to 96 samples in an ELISA format without laborious gel separation.
• Reliable: Shows high correlation with standard TRAP assay
• Safe: Nonradioactive method that can be performed in any laboratory
• Complete: All components necessary for elongation, amplification, and detection are provided in the kit

Assay time: Approximately 5 - 8 hours
Sample material: Cell or tissue extracts
Sensitivity: As sensitive as the radioactive (or isotopic) TRAP assay
Packaging
1 kit containing 15 components.
Principle
In detecting telomeric activity, a sample that contains telomerase will add these repetitive sequences to the 3′-end of the biotinylated synthetic P1-TS-primer (primer and nucleotides in Reaction mixture, bottle 2). In a second step, these elongated products are amplified by PCR using the primers P1-TS and P2, generating PCR amplicons. The Telomerase PCR ELISA combines both reactions in a one-step/ one-tube reaction. Optimized primer sequences eliminate the need for hot start PCR or the use of a wax barrier, and avoids amplification artifacts, such as primer dimers. An aliquot of the PCR is denatured and hybridized to a digoxigenin (DIG)-labeled, telomeric repeat-specific detection probe (contained in the Hybridization buffer, bottle 4). The hybridization products are immobilized via the biotin-labeled primer to a streptavidin-coated microplate. The immobilized PCR product is detected with an anti-digoxigenin antibody conjugated to peroxidase. The probe is finally visualized by peroxidase, which metabolizes TMB to form a colored reaction product. As an alternative to the ELISA protocol, the biotinylated primer may also be used for detection. If the telomerase-mediated six nucleotide incremental ladder is desired, fragments may be separated by PAGE, blotted onto a positively charged membrane, then detected appropriately (Biotin Luminescent Detection Kit).
Preparation Note
Working solution: Solution 5: Washing buffer, 1x
Dilute an appropriate volume of Washing buffer, 10x conc. with autoclaved double-dist. water (1:10) and mix thoroughly. Approx. 2.5 ml of the diluted Washing buffer are needed for one reaction.
Solution 6: Anti-DIG peroxidase, stock solution
Reconstitute the lyophilizate in 240 µl autoclaved double-dist. water. This results in an antibody conjugate concentration of 0.5 U/ml.
Solution 10: Positive control, cell extract
Reconstitute lyophilized cell extract on ice with 20 µl autoclaved double-dist. water (optionally treated with DEPC- or Velcorin) and mix thoroughly. The reconstituted solution has a concentration of about 1 x 103 cell equivalents per microliter.
Dispense solution into suitable aliquots (1 to 3 µl will be needed for 1 reaction).
Keep the extract on ice during pipetting.
Storage conditions (working solution): Washing buffer,1x:
Stable at 2 to 8 °C for 1month.
Anti-DIG peroxidase, stock solution:
Stable at 2 to 8 °C for 6 months.Do not freeze. Do not add sodium azid.
Anti-DIG peroxidase, working solution:
Prepare immediately before use. Do not store.
Positive control,cell extract:
The aliquoted cell extract is stable at -80 °C until the expiration date printed on the label. Avoid repeated freezing and thawing.
For life science research only. Not for use in diagnostic procedures.
Manufactured under license from Geron Corporation.
TeloTAGGG is a trademark of Roche

Quality Level	100,
usage	sufficient for 96 reactions
Торговая марка	Roche
shipped in	dry ice
storage temp.	20°C

pictograms	GHS05,GHS07
signalword	Warning
hcodes	H290 - H317
pcodes	P261 - P280 - P333 + P313 - P362 + P364 - P390 - P501
RIDADR	UN 3316 9
WGK Germany	WGK 2
Flash Point F	does not flash
Flash Point C	does not flash