Rat Mapk15 ELISA Kit (RTEB0582)- High Sensitivity

Product Name:	Rat Mitogen-activated protein kinase 15 (Mapk15) ELISA Kit
SKU:	RTEB0582
Size:	96T
Target:	Rat Mitogen-activated protein kinase 15 (Mapk15)
Synonyms:	Extracellular signal-regulated kinase 7, Extracellular signal-regulated kinase 8, ERK-7, ERK-8, MAP kinase 15, Erk7, Erk8
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Rat
Detection Range:	0.625-40ng/mL
Sensitivity:	0.299ng/mL

Intra CV:

4.1%

Inter CV:

9.6%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	109-121%	97-109%	91-104%	105-115%
EDTA Plasma(N=5)	109-119%	91-101%	92-101%	107-116%
Heparin Plasma(N=5)	85-97%	104-116%	109-119%	100-110%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	85	80-91
Plasma	87	81-93

Function:

Atypical MAPK protein that regulates several process such as autophagy, ciliogenesis, protein trafficking/secretion and genome integrity, in a kinase activity-dependent manner. Controls both, basal and starvation-induced autophagy throught its interaction with GABARAP, MAP1LC3B and GABARAPL1 leading to autophagosome formation, SQSTM1 degradation and reduced MAP1LC3B inhibitory phosphorylation. Regulates primary cilium formation and the localization of ciliary proteins involved in cilium structure, transport, and signaling. Prevents the relocation of the sugar-adding enzymes from the Golgi to the endoplasmic reticulum, thereby restricting the production of sugar-coated proteins. Upon amino-acid starvation, mediates transitional endoplasmic reticulum site disassembly and inhibition of secretion. Binds to chromatin leading to MAPK15 activation and interaction with PCNA, that which protects genomic integrity by inhibiting MDM2-mediated degradation of PCNA. Regulates DA transporter (DAT) activity and protein expression via activation of RhoA. In response to H(2)O(2) treatment phosphorylates ELAVL1, thus preventing it from binding to the PDCD4 3'UTR and rendering the PDCD4 mRNA accessible to miR-21 and leading to its degradation and loss of protein expression (By similarity). Also functions in a kinase activity-independent manner as a negative regulator of growth (PubMed:9891064). Phosphorylates in vitro FOS and MBP (PubMed:11875070). During oocyte maturation, plays a key role in the microtubule organization and mei- otic cell cycle progression in oocytes, fertilized eggs, and early embryos (By similarity). Interacts with ESRRA promoting its re-localization from the nucleus to the cytoplasm and then prevents its transcriptional activity (By similarity).

Uniprot:	Q9Z2A6
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant rat Mitogen-activated protein kinase 15
Sub Unit:	Interacts with CSK/c-Src, ABL1, RET and TGFB1I1. Interacts with GABARAP, MAP1LC3B and GABARAPL1; controls, in a kinase-dependent fashion, both basal and starvation-induced autophagy. Interacts with ESRRA; promotes re-localization of ESRRA to the cytoplasm through a XPO1-dependent mechanism then inhibits ESRRA transcriptional activity. Interacts with PCNA; the interaction is chromatin binding- and kinase activity-dependent and prevents MDM2-mediated PCNA destruction by inhibiting the association of PCNA with MDM2 (By similarity). Interacts with DVL2 (By similarity). Interacts with CLIC3; MAPK15 does not phosphorylates CLIC3 (PubMed:9880541).
Subcellular Location:	Cytoplasm Cytoskeleton Cilium basal body Cell junction Tight junction Cytoplasm Cytoskeleton Microtubule organizing center Centrosome Centriole Cytoplasmic vesicle Autophagosome Golgi apparatus Nucleus Cytoplasm Cytoplasm Cytoskeleton Spindle Co-localizes to the cytoplasm only in presence of ESRRA. Translocates to the nucleus upon activation (By similarity). At prometaphase I, metaphase I (MI), anaphase I, telophase I, and metaphase II (MII) stages, is stably detected at the spindle (By similarity).
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	Function: Constitutively active kinase which may function as a negative regulator of cell growth. In vitro, phosphorylates FOS.2 PublicationsManual assertion based on experiment in:Ref.1Catalytic activity: ATP + a protein = ADP + a phosphoprotein.Enzyme regulation: Activated by threonine and tyrosine phosphorylation. Inhibited by dual specificity phosphatases, such as DUSP1.
UniProt Protein Details:
NCBI Summary:	activity; localization, and function is regulated by the C-terminal tail [RGD, Feb 2006]
UniProt Code:	Q9Z2A6
NCBI GenInfo Identifier:	27545428
NCBI Gene ID:	286997
NCBI Accession:	NP_775453.1
UniProt Secondary Accession:	Q9Z2A6
UniProt Related Accession:	Q9Z2A6
Molecular Weight:	60,724 Da
NCBI Full Name:	mitogen-activated protein kinase 15
NCBI Synonym Full Names:	mitogen-activated protein kinase 15
NCBI Official Symbol:	Mapk15
NCBI Official Synonym Symbols:	Erk7
NCBI Protein Information:	mitogen-activated protein kinase 15; ERK-7; MAPK 15; MAP kinase 15; extracellular signal-regulated kinase 7
UniProt Protein Name:	Mitogen-activated protein kinase 15
UniProt Synonym Protein Names:	Extracellular signal-regulated kinase 7; ERK-7
Protein Family:	Mitogen-activated protein kinase
UniProt Gene Name:	Mapk15
UniProt Entry Name:	MK15_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.