Human STK ELISA Kit (HUFI04732)- High Sensitivity

Product Name:	Human STK (serine/threonine protein kinase) ELISA Kit
Product Code:	HUFI04732
Size:	96 Assays
Alias:	STK ELISA Kit
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human STK (serine/threonine protein kinase) concentrations in serum plasma and other biological fluids.
Sensitivity:	< 9.375ng/ml
Range:	15.625-1000ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human STK (serine/threonine protein kinase) and the recovery rates were calculated by comparing the measured value to the expected amount of Human STK (serine/threonine protein kinase) in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-105	97
EDTA plasma(n=5)	85-101	92
heparin plasma(n=5)	92-104	98

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human STK (serine/threonine protein kinase) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	89-100%	90-103%	88-105%
EDTA plasma(n=5)	83-98%	85-101%	85-101%
UFH plasma(n=5)	83-91%	81-100%	82-98%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

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

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

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 °C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37 °C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37 °C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37 °C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

Antibodies

Anti-AurkB Antibody (CAB1020)

Anti-Aurora B Antibody (CAB19539)

AURKB Antibody (PACO00424)

AURKB Antibody (PACO00425)

Phospho-AURKB (Y12) Antibody (PACO05719)