Cyclin H (Phospho-Thr315) Colorimetric Cell-Based ELISA Kit

Product Name:	Cyclin H (Phospho-Thr315) Colorimetric Cell-Based ELISA
Product Code:	CBCAB01407
ELISA Type:	Cell-Based
Target:	Cyclin H (Phospho-Thr315)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Cyclin H (Phospho-Thr315) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Cyclin H protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Cyclin H in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Cyclin H phosphorylation.

Qualitative determination of Cyclin H (Phospho-Thr315) concentration is achieved by an indirect ELISA format. In essence, Cyclin H (Phospho-Thr315) is captured by Cyclin H (Phospho-Thr315)-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: 902, UniProt ID: P51946, OMIM: 601953, Unigene: Hs.292524
Gene Symbol:	CCNH
Sub Type:	Phospho

UniProt Protein Function:	CCNH: Regulates CDK7, the catalytic subunit of the CDK- activating kinase (CAK) enzymatic complex. CAK activates the cyclin-associated kinases CDK1, CDK2, CDK4 and CDK6 by threonine phosphorylation. CAK complexed to the core-TFIIH basal transcription factor activates RNA polymerase II by serine phosphorylation of the repetitive C-terminus domain (CTD) of its large subunit (POLR2A), 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. Associates primarily with CDK7 and MAT1 to form the CAK complex. CAK can further associate with the core-TFIIH to form the TFIIH basal transcription factor. Belongs to the cyclin family. Cyclin C subfamily.
UniProt Protein Details:	Protein type:Cell cycle regulation Chromosomal Location of Human Ortholog: 5q13.3-q14 Cellular Component: nucleoplasm; holo TFIIH complex; nucleus; cyclin-dependent protein kinase activating kinase holoenzyme complex Molecular Function:RNA polymerase subunit kinase activity; DNA-dependent ATPase activity; protein binding; cyclin-dependent protein kinase regulator activity; protein kinase binding Biological Process: transcription from RNA polymerase II promoter; transcription initiation from RNA polymerase II promoter; viral reproduction; positive regulation of viral transcription; RNA elongation from RNA polymerase I promoter; transcription from RNA polymerase I promoter; termination of RNA polymerase I transcription; DNA repair; regulation of gene expression, epigenetic; protein amino acid phosphorylation; mRNA capping; negative regulation of gene expression, epigenetic; nucleotide-excision repair; transcription-coupled nucleotide-excision repair; RNA elongation from RNA polymerase II promoter; gene expression; positive regulation of transcription from RNA polymerase II promoter; nucleotide-excision repair, DNA damage removal; mitotic cell cycle; regulation of cyclin-dependent protein kinase activity; G2/M transition of mitotic cell cycle; transcription initiation from RNA polymerase I promoter; G1/S transition of mitotic cell cycle
NCBI Summary:	The protein encoded by this gene belongs to the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance through the cell cycle. Cyclins function as regulators of CDK kinases. Different cyclins exhibit distinct expression and degradation patterns which contribute to the temporal coordination of each mitotic event. This cyclin forms a complex with CDK7 kinase and ring finger protein MAT1. The kinase complex is able to phosphorylate CDK2 and CDC2 kinases, thus functions as a CDK-activating kinase (CAK). This cyclin and its kinase partner are components of TFIIH, as well as RNA polymerase II protein complexes. They participate in two different transcriptional regulation processes, suggesting an important link between basal transcription control and the cell cycle machinery. A pseudogene of this gene is found on chromosome 4. Alternate splicing results in multiple transcript variants.[provided by RefSeq, Nov 2010]
UniProt Code:	P51946
NCBI GenInfo Identifier:	1706232
NCBI Gene ID:	902
NCBI Accession:	P51946.1
UniProt Secondary Accession:	P51946,Q53X72, Q8TBL9,
UniProt Related Accession:	P51946
Molecular Weight:	323
NCBI Full Name:	Cyclin-H
NCBI Synonym Full Names:	cyclin H
NCBI Official Symbol:	CCNH
NCBI Official Synonym Symbols:	CAK; p34; p37; CycH
NCBI Protein Information:	cyclin-H; CAK complex subunit; MO15-associated protein; CDK-activating kinase complex subunit; cyclin-dependent kinase-activating kinase complex subunit
UniProt Protein Name:	Cyclin-H
UniProt Synonym Protein Names:	MO15-associated protein; p34; p37
Protein Family:	Cyclin
UniProt Gene Name:	CCNH
UniProt Entry Name:	CCNH_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-Cyclin H (Phospho-Thr315) Antibody, Anti-Cyclin H 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.)

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Cyclin H (Phospho-Thr315) Colorimetric Cell-Based ELISA Kit

Description