Human MAPK8 ELISA Kit (HUEB2610)- High Sensitivity

Product Name:	Human Mitogen-activated protein kinase 8 (MAPK8) ELISA Kit
SKU:	HUEB2610
Size:	96T
Target:	Human Mitogen-activated protein kinase 8 (MAPK8)
Synonyms:	JNK-46, Stress-activated protein kinase 1c, Stress-activated protein kinase JNK1, c-Jun N-terminal kinase 1, SAPK1c, MAP kinase 8, JNK1, PRKM8, SAPK1, SAPK1C
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.312-20ng/mL
Sensitivity:	0.2ng/mL

Intra CV:

5.6%

Inter CV:

10.9%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	111-120%	107-118%	99-109%	90-99%
EDTA Plasma(N=5)	95-105%	108-119%	109-119%	103-115%
Heparin Plasma(N=5)	102-114%	82-90%	81-91%	92-102%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	107	101-113
Plasma	109	103-115

Function:

JNK1 isoforms display different binding patterns: beta-1 preferentially binds to c-Jun, whereas alpha-1, alpha-2, and beta-2 have a similar low level of binding to both c-Jun or ATF2. However, there is no correlation between binding and phosphorylation, which is achieved at about the same efficiency by all isoforms.

Uniprot:	P45983
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Mitogen-activated protein kinase 8
Sub Unit:	Binds to at least four scaffolding proteins, MAPK8IP1/JIP-1, MAPK8IP2/JIP-2, MAPK8IP3/JIP-3/JSAP1 and SPAG9/MAPK8IP4/JIP-4. These proteins also bind other components of the JNK signaling pathway. Interacts with TP53 and WWOX. Interacts with JAMP. Forms a complex with MAPK8IP1 and ARHGEF28 (By similarity). Interacts with HSF1 (via D domain and preferentially with hyperphosphorylated form); this interaction occurs under both normal growth conditions and immediately upon heat shock (PubMed:10747973). Interacts (phosphorylated form) with NFE2; the interaction phosphorylates NFE2 in undifferentiated cells (By similarity). Interacts with NFATC4. Interacts with MECOM; regulates JNK signaling. Interacts with PIN1; this interaction mediates MAPK8 conformational changes leading to the binding of MAPK8 to its substrates.
Research Area:	Immunology
Subcellular Location:	Cytoplasm Nucleus
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.

ELISA

Human JNK ELISA Kit