Telomerase (Phospho-Ser824)Colorimetric Cell-Based ELISA Kit (CBCAB01477)

Product Name:	Telomerase (Phospho-Ser824)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01477
ELISA Type:	Cell-Based
Target:	Telomerase (Phospho-Ser824)
Reactivity:	Human
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Telomerase (Phospho-Ser824) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Telomerase protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Telomerase in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Telomerase phosphorylation.

Qualitative determination of Telomerase (Phospho-Ser824) concentration is achieved by an indirect ELISA format. In essence, Telomerase (Phospho-Ser824) is captured by Telomerase (Phospho-Ser824)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1.	A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2.	Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.

Database Information:	Gene ID: 7015, UniProt ID: O14746, OMIM: 127550/178500/187270/609135, Unigene: Hs.492203
Gene Symbol:	TERT
Sub Type:	Phospho

UniProt Protein Function:	TERT: telomerase reverse transcriptase is a ribonucleoprotein polymerase that maintains telomere ends by addition of the telomere repeat TTAGGG. The holoenzyme consists of TERT and an RNA component which serves as a template for the telomere repeat. Telomerase expression plays a role in cellular senescence, as it is normally repressed in postnatal somatic cells resulting in progressive shortening of telomeres. Deregulation of telomerase expression in somatic cells may be involved in oncogenesis. Studies in mouse suggest that telomerase also participates in chromosomal repair, since de novo synthesis of telomere repeats may occur at double-stranded breaks. Alternatively spliced isoforms have been identified, although the full-length sequence of some variants has not been determined.
UniProt Protein Details:	Protein type:Transferase; Nucleolus; EC 2.7.7.49; DNA-binding Chromosomal Location of Human Ortholog: 5p15.33 Cellular Component: nucleoplasm; PML body; chromosome, telomeric region; nuclear telomere cap complex; telomerase holoenzyme complex; nucleolus Molecular Function:telomerase activity; protein binding; protein homodimerization activity; RNA-directed RNA polymerase activity; DNA binding; metal ion binding; telomeric DNA binding; RNA-directed DNA polymerase activity; telomeric template RNA reverse transcriptase activity; nucleotidyltransferase activity; tRNA binding Biological Process: mitochondrion organization and biogenesis; DNA strand elongation; RNA-dependent DNA replication; positive regulation of nitric-oxide synthase activity; telomere maintenance via telomerase; age-dependent telomere shortening; positive regulation of Wnt receptor signaling pathway; RNA interference, production of siRNA; telomere maintenance Disease: Pulmonary Fibrosis, Idiopathic; Aplastic Anemia; Dyskeratosis Congenita, Autosomal Dominant, 1
NCBI Summary:	Telomerase is a ribonucleoprotein polymerase that maintains telomere ends by addition of the telomere repeat TTAGGG. The enzyme consists of a protein component with reverse transcriptase activity, encoded by this gene, and an RNA component which serves as a template for the telomere repeat. Telomerase expression plays a role in cellular senescence, as it is normally repressed in postnatal somatic cells resulting in progressive shortening of telomeres. Deregulation of telomerase expression in somatic cells may be involved in oncogenesis. Studies in mouse suggest that telomerase also participates in chromosomal repair, since de novo synthesis of telomere repeats may occur at double-stranded breaks. Alternatively spliced variants encoding different isoforms of telomerase reverse transcriptase have been identified; the full-length sequence of some variants has not been determined. Alternative splicing at this locus is thought to be one mechanism of regulation of telomerase activity. [provided by RefSeq, Jul 2008]
UniProt Code:	O14746
NCBI GenInfo Identifier:	6226780
NCBI Gene ID:	7015
NCBI Accession:	O14746.1
UniProt Secondary Accession:	O14746,O14783, Q2XS35, Q8N6C3, Q8NG38, Q8NG46,
UniProt Related Accession:	O14746
Molecular Weight:	88,965 Da
NCBI Full Name:	Telomerase reverse transcriptase
NCBI Synonym Full Names:	telomerase reverse transcriptase
NCBI Official Symbol:	TERT
NCBI Official Synonym Symbols:	TP2; TRT; CMM9; EST2; TCS1; hTRT; DKCA2; DKCB4; hEST2; PFBMFT1
NCBI Protein Information:	telomerase reverse transcriptase; telomerase catalytic subunit; telomerase-associated protein 2
UniProt Protein Name:	Telomerase reverse transcriptase
UniProt Synonym Protein Names:	HEST2; Telomerase catalytic subunit; Telomerase-associated protein 2; TP2
Protein Family:	Telomerase reverse transcriptase
UniProt Gene Name:	TERT
UniProt Entry Name:	TERT_HUMAN

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent papers or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 ml
Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
Orbital shaker (optional)
Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1.	Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37 °C prior to adding cells.
2.	Incubate the cells for overnight at 37 °C, 5% CO2.
3.	Treat the cells as desired.
4.	Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.	Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.	Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4 °C for a week.
7.	Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.	Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.	Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.	Add 50 µL of 1x primary antibodies Anti-Telomerase (Phospho-Ser824) Antibody, Anti-Telomerase Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4 °C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.	Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.	Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.	Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)