Rat Mitogen-activated protein kinase 14 (Mapk14) ELISA Kit

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SKU:
RTEB1632
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P70618
ELISA Type:
Sandwich
Synonyms:
MAPK
Reactivity:
Rat
Frequently bought together:

Description

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Product Name:Rat Mitogen-activated protein kinase 14 (Mapk14) ELISA Kit
Product Code:RTEB1632
Alias:Mitogen-activated protein kinase 14, MAP kinase 14, MAPK 14, CRK1, Mitogen-activated protein kinase p38 alpha, MAP kinase p38 alpha, Mapk14, Csbp1, Csbp2, 2.7.11.24
Uniprot:P70618
Reactivity:Rat
Range:Please contact us for more information
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:P38A: a proline-directed ser/thr MAP kinase, and one of four p38 kinases that play important roles in cellular responses to inflammatory cytokines, DNA damage, oxidative stress, and some GPCRs, leading to direct activation of transcription factors and of other downstream kinases including MSK1, MSK2, eEF2K, MK2, and PRAK. MSK1 and -2 play important roles in the rapid induction of immediate-early genes in response to stress or mitogenic stimuli. MK2 and -3 control gene expression mostly at the post-transcriptional level. eEF2K is important for the elongation of mRNA during translation. Ectodomain shedding of transmembrane proteins is regulated by p38 MAPKs as well. In response to inflammatory stimuli, p38 MAPKs phosphorylate the membrane-associated metalloprotease ADAM17, which then cleaves the ectodomain of TGF-alpha family ligands, a process leading to the activation of EGFR signaling and cell proliferation. In the nucleus, many transcription factors are phosphorylated and activated by p38 MAPKs in response to different stimuli. Classical examples include ATF1, ATF2, ATF6, ELK1, PTPRH, CHOPO, p53 and MEF2C and MEF2A. The p38 MAPKs are emerging as important modulators of gene expression by regulating chromatin modifiers and remodelers. The promoters of several genes involved in the inflammatory response, such as IL6, IL8 and IL12B, display a p38 MAPK-dependent enrichment of histone H3 phosphorylation on 'Ser-10' (H3S10ph) in LPS-stimulated myeloid cells. Interacts directly with HDAC3 interacts directly and selectively to repress ATF2 transcriptional activity, and regulate TNF gene expression in LPS-stimulated cells. Phosphorylates the ubiquitin ligase SIAH2, regulating its activity towards EGLN3. May also inhibit the lysosomal degradation pathway of autophagy by interfering with the intracellular trafficking of the transmembrane protein ATG9. Regulates the endocytosis of membrane receptors that depend on RAB5A. Regulates the clathrin-mediated internalization of EGFR induced by inflammatory cytokines and UV irradiation by phosphorylating the EGFR and RAB5A effectors. Required in mid-fetal development for the growth of embryo-derived blood vessels in the labyrinth layer of the placenta. Plays an essential role in developmental and stress-induced erythropoiesis, through regulation of EPO gene expression. Interacts with casein kinase II subunits CSNK2A1 and CSNK2B. Activated by cell stresses such as DNA damage, heat shock, osmotic shock, anisomycin and sodium arsenite, as well as pro-inflammatory stimuli such as LPS and IL-1. Phosphorylated by ZAP70 in an alternative activation pathway in response to TCR signaling in T-cells, a pathway is inhibited by GADD45A. Four alternatively spliced isoforms of the human protein have been observed. Isoform MXI2 activation is stimulated by mitogens and oxidative stress and only poorly phosphorylates ELK1 and ATF2. Isoform EXIP may play a role in the early onset of apoptosisProtein type: Protein kinase, CMGC; EC 2.7.11.24; Kinase, protein; Protein kinase, Ser/Thr (non-receptor); CMGC group; MAPK family; p38 subfamily; MAPK/p38 subfamilyCellular Component: nucleoplasm; spindle pole; mitochondrion; cell; cytoplasm; nucleus; cytosolMolecular Function: protein C-terminus binding; MAP kinase activity; protein serine/threonine kinase activity; protein binding; NFAT protein binding; kinase activity; ATP binding; protein kinase activityBiological Process: apoptosis; protein amino acid autophosphorylation; stress-activated MAPK cascade; cell morphogenesis; response to lipopolysaccharide; osteoclast differentiation; protein amino acid phosphorylation; positive regulation of glucose import; regulation of transcription, DNA-dependent; transmembrane receptor protein serine/threonine kinase signaling pathway; response to glucose stimulus; lipopolysaccharide-mediated signaling pathway; chondrocyte differentiation; angiogenesis; placenta development; cartilage condensation; positive regulation of cardiac muscle cell proliferation; skeletal muscle development; transcription, DNA-dependent; positive regulation of erythrocyte differentiation; positive regulation of blood vessel endothelial cell migration; MAPKKK cascade; DNA damage response, signal transduction; glucose metabolic process; positive regulation of myoblast differentiation; regulation of transcription from RNA polymerase II promoter; peptidyl-serine phosphorylation; positive regulation of protein import into nucleus; response to muramyl dipeptide; DNA damage checkpoint; striated muscle cell differentiation; positive regulation of transcription from RNA polymerase II promoter; fatty acid oxidation; vascular endothelial growth factor receptor signaling pathway; response to DNA damage stimulus; myoblast cell differentiation involved in skeletal muscle regeneration
UniProt Protein Details:
NCBI Summary:mitogen-activated protein kinase; involved in intracellular signalling, inhibition of apoptosis and gene activation [RGD, Feb 2006]
UniProt Code:P70618
NCBI GenInfo Identifier:2499601
NCBI Gene ID:81649
NCBI Accession:P70618.3
UniProt Secondary Accession:P70618,O08594, Q99MG4
UniProt Related Accession:P70618
Molecular Weight:41,521 Da
NCBI Full Name:Mitogen-activated protein kinase 14
NCBI Synonym Full Names:mitogen activated protein kinase 14
NCBI Official Symbol:Mapk14  
NCBI Official Synonym Symbols:RK; Hog; p38; CRK1; CSBP; Exip; Mxi2; CSPB1; Csbp1; Csbp2; Prkm14; Prkm15; Sapk2A; p38Hog; p38alpha  
NCBI Protein Information:mitogen-activated protein kinase 14; Csaids binding protein; MAP kinase 14; MAP kinase 2; MAP kinase Mxi2; MAP kinase p38 alpha; MAPK 14; MAX-interacting protein 2; cytokine suppressive anti-inflammatory drug binding protein; cytokine-supressive anti-inflammatory drug binding protein; mitogen-activated protein kinase 14A; mitogen-activated protein kinase p38 alpha; p38 MAP kinase; p38 mitogen activated protein kinase; p38alpha Exip; reactive kinase; stress-activated protein kinase 2A
UniProt Protein Name:Mitogen-activated protein kinase 14
UniProt Synonym Protein Names:CRK1; Mitogen-activated protein kinase p38 alpha; MAP kinase p38 alpha
Protein Family:
UniProt Gene Name:Mapk14  
UniProt Entry Name:MK14_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.

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