Human JNK ELISA Kit (HUFI02082)- High Sensitivity

Product Name:	Human JNK ELISA Kit
Product Code:	HUFI02082
Size:	96 Assays
Alias:	MAPK8, PRKM8, SAPK1, c-Jun N-terminal kinase 1, JNK1 alpha protein kinase, JNK1 beta protein kinase, JNK1JNK1A2, JNK-46, JUN N-terminal kinase, MAP kinase 8, MAPK 8, mitogen-activated protein kinase 8, mitogen-activated protein kinase 8 isoform JNK1 alpha1, mitogen-activated protein kinase 8 isoform JNK1 beta2, PRKM8JNK, protein kinase JNK1, SAPK1JNK21B1, 2, Stress-activated protein kinase 1, Stress-activated protein kinase JNK1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human MAPK8 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.188ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human MAPK8 and the recovery rates were calculated by comparing the measured value to the expected amount of Human MAPK8 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	86-102	94
EDTA plasma(n=5)	88-98	93
UFH plasma(n=5)	85-105	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human MAPK8 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)	86-97%	93-104%	95-102%
EDTA plasma(n=5)	82-92%	83-100%	85-101%
UFH plasma(n=5)	83-100%	82-98%	86-97%

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

P45983

UniProt Protein Function:	JNK1: a protein kinase of the MAPK family that is potently activated by a variety of environmental stresses, including UV and gamma radiation, ceramides, pro-inflammatory cytokines and, in some instances, by growth factors and GPCR agonists. Substrates include a number of transcription factors, primarily components of AP-1 such as c-Jun and ATF2, thus regulating AP-1 transcriptional activity. In T- cells, JNK1 and JNK2 are required for polarized differentiation of T-helper cells into Th1 cells. Binds to at least four scaffolding proteins, JIP-1, -2, -3 and -4. Activity increased in obesity. Inhibition or mouse knockout increases insulin sensitivity. Part of NFB pathway involved in inflammation and cancer, and signals downstream of Ras, though possibly as an apoptotic negative regulator of growth. Four alternatively-spliced isoforms have been described.
UniProt Protein Details:	Protein type:Kinase, protein; EC 2.7.11.24; Protein kinase, CMGC; Protein kinase, Ser/Thr (non-receptor); CMGC group; MAPK family; MAPK/JNK subfamily; JNK subfamily Chromosomal Location of Human Ortholog: 10q11.22 Cellular Component: nucleoplasm; mitochondrion; cytosol; nucleus Molecular Function:protein serine/threonine kinase activity; protein binding; enzyme binding; histone deacetylase regulator activity; histone deacetylase binding; JUN kinase activity; ATP binding Biological Process: nerve growth factor receptor signaling pathway; apoptosis; positive regulation of apoptosis; rhythmic process; stress-activated MAPK cascade; toll-like receptor 3 signaling pathway; protein amino acid phosphorylation; toll-like receptor 10 signaling pathway; toll-like receptor 5 signaling pathway; regulation of protein localization; regulation of transcription factor activity; response to stress; JNK cascade; negative regulation of protein binding; toll-like receptor 4 signaling pathway; response to UV; ossification; regulation of histone deacetylation; MyD88-independent toll-like receptor signaling pathway; peptidyl-threonine phosphorylation; regulation of circadian rhythm; JUN phosphorylation; toll-like receptor 2 signaling pathway; MyD88-dependent toll-like receptor signaling pathway; peptidyl-serine phosphorylation; response to cadmium ion; toll-like receptor signaling pathway; innate immune response; toll-like receptor 9 signaling pathway; negative regulation of apoptosis
NCBI Summary:	The protein encoded by this gene is a member of the MAP kinase family. MAP kinases act as an integration point for multiple biochemical signals, and are involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development. This kinase is activated by various cell stimuli, and targets specific transcription factors, and thus mediates immediate-early gene expression in response to cell stimuli. The activation of this kinase by tumor-necrosis factor alpha (TNF-alpha) is found to be required for TNF-alpha induced apoptosis. This kinase is also involved in UV radiation induced apoptosis, which is thought to be related to cytochrom c-mediated cell death pathway. Studies of the mouse counterpart of this gene suggested that this kinase play a key role in T cell proliferation, apoptosis and differentiation. Five alternatively spliced transcript variants encoding distinct isoforms have been reported. [provided by RefSeq, Jun 2013]
UniProt Code:	P45983
NCBI GenInfo Identifier:	2507195
NCBI Gene ID:	5599
NCBI Accession:	P45983.2
UniProt Secondary Accession:	P45983,Q15709, Q15712, Q15713, Q308M2, B5BTZ5, B7ZLV4 D3DX88, D3DX92,
UniProt Related Accession:	P45983
Molecular Weight:	427
NCBI Full Name:	Mitogen-activated protein kinase 8
NCBI Synonym Full Names:	mitogen-activated protein kinase 8
NCBI Official Symbol:	MAPK8
NCBI Official Synonym Symbols:	JNK; JNK1; PRKM8; SAPK1; JNK-46; JNK1A2; SAPK1c; JNK21B1/2
NCBI Protein Information:	mitogen-activated protein kinase 8; MAP kinase 8; JUN N-terminal kinase; c-Jun N-terminal kinase 1; stress-activated protein kinase 1; stress-activated protein kinase 1c; mitogen-activated protein kinase 8 isoform JNK1 beta2; mitogen-activated protein kin
UniProt Protein Name:	Mitogen-activated protein kinase 8
UniProt Synonym Protein Names:	JNK-46; Stress-activated protein kinase 1c; SAPK1c; Stress-activated protein kinase JNK1; c-Jun N-terminal kinase 1
Protein Family:	JNK-interacting protein
UniProt Gene Name:	MAPK8
UniProt Entry Name:	MK08_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.