CDK7 (Phospho-Thr170)Colorimetric Cell-Based ELISA Kit (CBCAB01403)

Product Name:	CDK7 (Phospho-Thr170)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01403
ELISA Type:	Cell-Based
Target:	CDK7 (Phospho-Thr170)
Reactivity:	Human, Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The CDK7 (Phospho-Thr170) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CDK7 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CDK7 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CDK7 phosphorylation.

Qualitative determination of CDK7 (Phospho-Thr170) concentration is achieved by an indirect ELISA format. In essence, CDK7 (Phospho-Thr170) is captured by CDK7 (Phospho-Thr170)-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: 1022, UniProt ID: P50613, OMIM: 601955, Unigene: Hs.184298
Gene Symbol:	CDK7
Sub Type:	Phospho

UniProt Protein Function:	CDK7: a protein kinase of the CDK family. Forms a trimeric complex with cyclin H and MAT1, which functions as a Cdk-activating kinase (CAK). Activates the cyclin-associated kinases CDK1, -2, -4 and -6. An essential component of the transcription factor TFIIH, that is involved in transcription initiation and DNA repair. Serves as a direct link between the regulation of transcription and the cell cycle. Phosphorylates and activates RNA polymerase II, allowing its escape from the promoter and elongation of the transcripts. Involved in cell cycle control and in RNA transcription by RNA polymerase II. Its expression and activity are constant throughout the cell cycle.
UniProt Protein Details:	Protein type:Protein kinase, CMGC; Nuclear receptor co-regulator; Protein kinase, Ser/Thr (non-receptor); Kinase, protein; Cell cycle regulation; EC 2.7.11.23; EC 2.7.11.22; CMGC group; CDK family; CDK7 subfamily; CDK/CDK7 subfamily Chromosomal Location of Human Ortholog: 5q12.1 Cellular Component: cytoplasm; holo TFIIH complex; mitochondrion; nucleoplasm; nucleus Molecular Function:DNA-dependent ATPase activity; kinase activity; protein binding; protein C-terminus binding; protein kinase activity; protein serine/threonine kinase activity; RNA polymerase subunit kinase activity Biological Process: cell cycle arrest; cell proliferation; G1/S transition of mitotic cell cycle; G2/M transition of mitotic cell cycle; mRNA capping; nucleotide-excision repair, preincision complex assembly; positive regulation of transcription from RNA polymerase II promoter; regulation of cyclin-dependent protein kinase activity; RNA elongation from RNA polymerase I promoter; RNA elongation from RNA polymerase II promoter; snRNA transcription from RNA polymerase II promoter; termination of RNA polymerase I transcription; transcription from RNA polymerase II promoter; transcription initiation from RNA polymerase I promoter; transcription initiation from RNA polymerase II promoter; transcription-coupled nucleotide-excision repair
NCBI Summary:	The protein encoded by this gene is a member of the cyclin-dependent protein kinase (CDK) family. CDK family members are highly similar to the gene products of Saccharomyces cerevisiae cdc28, and Schizosaccharomyces pombe cdc2, and are known to be important regulators of cell cycle progression. This protein forms a trimeric complex with cyclin H and MAT1, which functions as a Cdk-activating kinase (CAK). It is an essential component of the transcription factor TFIIH, that is involved in transcription initiation and DNA repair. This protein is thought to serve as a direct link between the regulation of transcription and the cell cycle. [provided by RefSeq, Jul 2008]
UniProt Code:	P50613
NCBI GenInfo Identifier:	1705722
NCBI Gene ID:	1022
NCBI Accession:	P50613.1
UniProt Secondary Accession:	P50613,Q9BS60, Q9UE19,
UniProt Related Accession:	P50613
Molecular Weight:	39,038 Da
NCBI Full Name:	Cyclin-dependent kinase 7
NCBI Synonym Full Names:	cyclin dependent kinase 7
NCBI Official Symbol:	CDK7
NCBI Official Synonym Symbols:	CAK; CAK1; HCAK; MO15; STK1; CDKN7; p39MO15
NCBI Protein Information:	cyclin-dependent kinase 7
UniProt Protein Name:	Cyclin-dependent kinase 7
UniProt Synonym Protein Names:	39 kDa protein kinase; p39 Mo15; CDK-activating kinase 1; Cell division protein kinase 7; Serine/threonine-protein kinase 1; TFIIH basal transcription factor complex kinase subunit
Protein Family:	Cyclin-dependent kinase
UniProt Gene Name:	CDK7
UniProt Entry Name:	CDK7_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-CDK7 (Phospho-Thr170) Antibody, Anti-CDK7 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.)