Product Name:	Rat Mitogen-activated protein kinase 3 (Mapk3) ELISA Kit
SKU:	RTEB0908
Size:	96T
Target:	Rat Mitogen-activated protein kinase 3 (Mapk3)
Synonyms:	ERT2, Extracellular signal-regulated kinase 1, Insulin-stimulated MAP2 kinase, MAP kinase isoform p44, MNK1, Microtubule-associated protein 2 kinase, p44-ERK1, ERK-1, p44-MAPK, MAP kinase 3, Erk1, Prkm3
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Rat
Detection Range:	0.156-10ng/mL
Sensitivity:	0.09ng/mL

Intra CV:

5.8%

Inter CV:

9.3%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	104-114%	104-112%	106-116%	109-118%
EDTA Plasma(N=5)	101-101%	93-104%	89-99%	88-101%
Heparin Plasma(N=5)	107-117%	91-101%	99-109%	80-89%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	106	100-112
Plasma	108	102-114

Function:

Serine/threonine kinase which acts as an essential component of the MAP kinase signal transduction pathway. MAPK1/ERK2 and MAPK3/ERK1 are the 2 MAPKs which play an important role in the MAPK/ERK cascade. They participate also in a signaling cascade initiated by activated KIT and KITLG/SCF. Depending on the cellular context, the MAPK/ERK cascade mediates diverse biological functions such as cell growth, adhesion, survival and differentiation through the regulation of transcription, translation, cytoskeletal rearrangements. The MAPK/ERK cascade plays also a role in initiation and regulation of meiosis, mitosis, and postmitotic functions in differentiated cells by phosphorylating a number of transcription factors. About 160 substrates have already been discovered for ERKs. Many of these substrates are localized in the nucleus, and seem to participate in the regulation of transcription upon stimulation. However, other substrates are found in the cytosol as well as in other cellular organelles, and those are responsible for processes such as translation, mitosis and apoptosis. Moreover, the MAPK/ERK cascade is also involved in the regulation of the endosomal dynamics, including lysosome processing and endosome cycling through the perinuclear recycling compartment (PNRC); as well as in the fragmentation of the Golgi apparatus during mitosis. The substrates include transcription factors (such as ATF2, BCL6, ELK1, ERF, FOS, HSF4 or SPZ1), cytoskeletal elements (such as CANX, CTTN, GJA1, MAP2, MAPT, PXN, SORBS3 or STMN1), regulators of apoptosis (such as BAD, BTG2, CASP9, DAPK1, IER3, MCL1 or PPARG), regulators of translation (such as EIF4EBP1) and a variety of other signaling-related molecules (like ARHGEF2, FRS2 or GRB10). Protein kinases (such as RAF1, RPS6KA1/RSK1, RPS6KA3/RSK2, RPS6KA2/RSK3, RPS6KA6/RSK4, SYK, MKNK1/MNK1, MKNK2/MNK2, RPS6KA5/MSK1, RPS6KA4/MSK2, MAPKAPK3 or MAPKAPK5) and phosphatases (such as DUSP1, DUSP4, DUSP6 or DUSP16) are other substrates which enable the propagation the MAPK/ERK signal to additional cytosolic and nuclear targets, thereby extending the specificity of the cascade.

Uniprot:	P21708
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant rat Mitogen-activated protein kinase 3
Sub Unit:	Binds both upstream activators and downstream substrates in multimolecular complexes. Found in a complex with at least BRAF, HRAS, MAP2K1/MEK1, MAPK3 and RGS14. Interacts with ADAM15, ARRB2, CANX, DAPK1 (via death domain), HSF4, IER3, MAP2K1/MEK1, MORG1, NISCH, PEA15, SGK1 and MKNK2 (By similarity). MKNK2 isoform 1 binding prevents from dephosphorylation and inactivation. Interacts with TPR (By similarity). Interacts with HSF1 (via D domain and preferentially with hyperphosphorylated form); this interaction occurs upon heat shock (By similarity). Interacts with CDKN2AIP.
Research Area:	Stem Cells
Subcellular Location:	Isoform 2 Nucleus
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	ERK1: a serine/threonine kinase of the GMGC group that plays a critical role in the regulation of cell growth and differentiation. ERK1 (MAPK3) and ERK2 (MAPK1) play central roles in MAPK cascades and are activated by a wide variety of extracellular signals including growth and neurotrophic factors, cytokines, hormones and neurotransmitters. Depending on the cellular context, MAPK cascades mediate diverse biological functions such as cell growth, adhesion, survival and differentiation through the regulation of transcription, translation, cytoskeletal rearrangements. MAPK cascades also plays a role in initiation and regulation of meiosis, mitosis, and postmitotic functions in differentiated cells by phosphorylating a number of transcription factors. Activation of MAP kinases occurs through phosphorylation of threonine and tyrosine residues at the sequence TEY by upstream MAP kinase kinases, MEK1 and -2. Phosphorylation of both the threonine and tyrosine are required for activity. This phosphorylation causes dramatic conformational changes, which enable full activation and interaction of MAPK1/ERK2 with its substrates.
UniProt Protein Details:	Protein type:Protein kinase, Ser/Thr (non-receptor); Protein kinase, CMGC; Kinase, protein; EC 2.7.11.24; CMGC group; MAPK family; MAPK/ERK subfamily; ERK subfamily Cellular Component: caveola; cytoplasm; cytoskeleton; cytosol; early endosome; focal adhesion; Golgi apparatus; late endosome; microtubule cytoskeleton; mitochondrion; nuclear envelope; nucleoplasm; nucleus; protein complex; pseudopodium Molecular Function:ATP binding; MAP kinase activity; phosphatase binding; phosphotyrosine binding; protein binding; protein kinase activity; protein serine/threonine kinase activity Biological Process: apoptosis; arachidonic acid metabolic process; Bergmann glial cell differentiation; BMP signaling pathway; cartilage development; DNA damage induced protein phosphorylation; lipopolysaccharide-mediated signaling pathway; MAPKKK cascade; neural crest cell development; nuclear translocation of MAPK; organ morphogenesis; outer ear morphogenesis; peptidyl-serine phosphorylation; phosphorylation; positive regulation of cyclase activity; positive regulation of histone acetylation; positive regulation of histone phosphorylation; positive regulation of protein amino acid phosphorylation; positive regulation of telomerase activity; positive regulation of telomere maintenance via telomerase; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; positive regulation of translation; protein amino acid phosphorylation; protein complex assembly; regulation of cytoskeleton organization and biogenesis; regulation of ossification; regulation of stress-activated MAPK cascade; regulation of transcription factor activity; response to DNA damage stimulus; response to exogenous dsRNA; response to lipopolysaccharide; response to toxin; sensory perception of pain; thymus development; thyroid gland development; transcription, DNA-dependent
NCBI Summary:	kinase involved in intracellular signalling; component of Mapk signalling pathway [RGD, Feb 2006]
UniProt Code:	P21708
NCBI GenInfo Identifier:	115311606
NCBI Gene ID:	50689
NCBI Accession:	P21708.5
UniProt Secondary Accession:	P21708,Q4PIY8, Q62686, Q9JJ13,
UniProt Related Accession:	P21708
Molecular Weight:	45,770 Da
NCBI Full Name:	Mitogen-activated protein kinase 3
NCBI Synonym Full Names:	mitogen activated protein kinase 3
NCBI Official Symbol:	Mapk3
NCBI Official Synonym Symbols:	p44; ERK1; ERT2; MNK1; Erk-1; Esrk1; MAPK1; Prkm3; p44erk1; p44mapk
NCBI Protein Information:	mitogen-activated protein kinase 3
UniProt Protein Name:	Mitogen-activated protein kinase 3
UniProt Synonym Protein Names:	ERT2; Extracellular signal-regulated kinase 1; ERK-1; Insulin-stimulated MAP2 kinase; MAP kinase isoform p44; p44-MAPK; MNK1; Microtubule-associated protein 2 kinase; p44-ERK1
UniProt Gene Name:	Mapk3
UniProt Entry Name:	MK03_RAT

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.

Rat Mitogen-activated protein kinase 3 (Mapk3) ELISA Kit (RTEB0908)

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