AurB (Phospho-Thr232)Colorimetric Cell-Based ELISA Kit (CBCAB00449)

Product Name:	AurB (Phospho-Thr232)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00449
ELISA Type:	Cell-Based
Target:	AurB (Phospho-Thr232)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The AurB (Phospho-Thr232) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect AurB protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated AurB in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on AurB phosphorylation.

Qualitative determination of AurB (Phospho-Thr232) concentration is achieved by an indirect ELISA format. In essence, AurB (Phospho-Thr232) is captured by AurB (Phospho-Thr232)-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary 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: 9212, UniProt ID: Q96GD4, OMIM: 604970, Unigene: Hs.442658
Gene Symbol:	AURKB
Sub Type:	Phospho

UniProt Protein Function:	AurB: a member of the AUR family of kinases. May be directly involved in regulating the cleavage of polar spindle microtubules and is a key regulator for the onset of cytokinesis during mitosis. Expressed during S and G2/M phase and expression is upregulated in cancer cells during M phase. Localized to the midzone of central spindle in late anaphase and concentrated into the midbody in telophase and cytokinesis. Colocalizes with gamma tubulin in the mid-body. Component of the chromosomal passenger complex (CPC), a complex that acts as a key regulator of mitosis. The CPC complex has essential functions at the centromere in ensuring correct chromosome alignment and segregation and is required for chromatin-induced microtubule stabilization and spindle assembly. Overexpressed in colorectal and other cancer cell lines and thought to cause aneuploidy via histone phosphorylation.
UniProt Protein Details:	Protein type:Protein kinase, Ser/Thr (non-receptor); EC 2.7.11.1; Kinase, protein; Protein kinase, Other; Other group; AUR family Chromosomal Location of Human Ortholog: 17p13.1 Cellular Component: chromocenter; condensed nuclear chromosome, pericentric region; cytosol; intercellular bridge; kinetochore; midbody; nucleoplasm; nucleus; spindle; spindle microtubule; spindle midzone; spindle pole centrosome Molecular Function:ATP binding; histone serine kinase activity; metal ion binding; protein binding; protein serine/threonine kinase activity; protein serine/threonine/tyrosine kinase activity Biological Process: abscission; aging; anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolic process; attachment of spindle microtubules to kinetochore; cell proliferation; cellular protein metabolic process; histone modification; mitotic cell cycle; negative regulation of B cell apoptosis; negative regulation of cytokinesis; negative regulation of protein binding; negative regulation of transcription from RNA polymerase II promoter; positive regulation of cytokinesis; positive regulation of telomerase activity; positive regulation of telomere maintenance via telomerase; post-translational protein modification; protein amino acid autophosphorylation; protein amino acid phosphorylation; protein sumoylation; regulation of chromosome segregation; small GTPase mediated signal transduction; spindle checkpoint; spindle midzone assembly involved in mitosis; spindle stabilization
NCBI Summary:	This gene encodes a member of the aurora kinase subfamily of serine/threonine kinases. The genes encoding the other two members of this subfamily are located on chromosomes 19 and 20. These kinases participate in the regulation of alignment and segregation of chromosomes during mitosis and meiosis through association with microtubules. A pseudogene of this gene is located on chromosome 8. Alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Sep 2015]
UniProt Code:	Q96GD4
NCBI GenInfo Identifier:	317373473
NCBI Gene ID:	9212
NCBI Accession:	Q96GD4.3
UniProt Secondary Accession:	Q96GD4,O14630, O60446, O95083, Q96DV5, Q9UQ46, B4DNM4 C7G533, C7G534, C7G535, D3DTR4, J9JID1,
UniProt Related Accession:	Q96GD4
Molecular Weight:	39,467 Da
NCBI Full Name:	Aurora kinase B
NCBI Synonym Full Names:	aurora kinase B
NCBI Official Symbol:	AURKB
NCBI Official Synonym Symbols:	AIK2; AIM1; ARK2; AurB; IPL1; STK5; AIM-1; STK12; PPP1R48; aurkb-sv1; aurkb-sv2
NCBI Protein Information:	aurora kinase B
UniProt Protein Name:	Aurora kinase B
UniProt Synonym Protein Names:	Aurora 1; Aurora- and IPL1-like midbody-associated protein 1; AIM-1; Aurora/IPL1-related kinase 2; ARK-2; Aurora-related kinase 2; STK-1; Serine/threonine-protein kinase 12; Serine/threonine-protein kinase 5; Serine/threonine-protein kinase aurora-B
Protein Family:	Aurora kinase
UniProt Gene Name:	AURKB
UniProt Entry Name:	AURKB_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-AurB (Phospho-Thr232) Antibody, Anti-AurB 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.)