Bovine Mitogen-activated protein kinase 1 (MAPK1) ELISA Kit
The Bovine Mitogen-Activated Protein Kinase 1 (MAPK1) ELISA Kit is a powerful tool for detecting and quantifying MAPK1 levels in bovine samples such as serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit delivers accurate and reproducible results, making it ideal for various research applications.MAPK1 is a key player in cellular signaling pathways and is involved in regulating processes such as cell growth, differentiation, and apoptosis.
Dysregulation of MAPK1 has been linked to various diseases, including cancer, inflammatory disorders, and neurodegenerative conditions, highlighting its importance as a potential therapeutic target and biomarker. By using the Bovine MAPK1 ELISA Kit, researchers can gain valuable insights into the role of MAPK1 in bovine physiology and pathology, paving the way for innovative research and drug development strategies.
Product Name:
Bovine Mitogen-activated protein kinase 1 (MAPK1) ELISA Kit
Acts as a transcriptional repressor. Binds to a [GC]AAA[GC] consensus sequence. Repress the expression of interferon gamma-induced genes. Seems to bind to the promoter of CCL5, DMP1, IFIH1, IFITM1, IRF7, IRF9, LAMP3, OAS1, OAS2, OAS3 and STAT1. Transcriptional activity is independent of kinase activity.
Uniprot:
P46196
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant bovine Mitogen-activated protein kinase 1
Sub Unit:
Binds both upstream activators and downstream substrates in multimolecular complexes. Interacts with ADAM15, ARHGEF2, ARRB2, DAPK1 (via death domain), HSF4, IER3, IPO7, MKNK2, DUSP6, MORG1, NISCH, PEA15, SGK1, and isoform 1 of NEK2 (By similarity). Interacts (phosphorylated form) with CAV2 ('Tyr-19'-phosphorylated form); the interaction, promoted by insulin, leads to nuclear location and MAPK1 activation (By similarity). MKNK2 isoform 1 binding prevents from dephosphorylation and inactivation (By similarity). Interacts with DCC (By similarity). The phosphorylated form interacts with PML (By similarity). Interacts with STYX. Interacts with CDK2AP2.
Research Area:
Neurosciences
Subcellular Location:
Nucleus Cytoplasm Cytoskeleton Microtubule organizing center Centrosome Cytoplasm Cytoplasm Cytoskeleton Spindle PEA15-binding and phosphorylated DAPK1 promote its cytoplasmic retention. Phosphorylation at Ser-246 and Ser-248 as well as autophosphorylation at Thr-190 promote nuclear localization. Associated with the spindle during prometaphase and metaphase.
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
ERK2: 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 T*EY* 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.Protein type: Protein kinase, Ser/Thr (non-receptor); Kinase, protein; Protein kinase, CMGC; EC 2.7.11.24; CMGC group; MAPK family; MAPK/ERK subfamily; ERK subfamilyChromosomal Location of Human Ortholog: 22q11.21Cellular Component: dendrite cytoplasm; Golgi apparatus; protein complex; focal adhesion; mitochondrion; early endosome; perikaryon; pseudopodium; caveola; cytosol; nucleoplasm; microtubule cytoskeleton; cytoskeleton; axon; late endosome; cytoplasm; microtubule organizing center; nucleusMolecular Function: RNA polymerase subunit kinase activity; MAP kinase activity; protein serine/threonine kinase activity; protein binding; DNA binding; phosphotyrosine binding; mitogen-activated protein kinase kinase kinase binding; transcription factor binding; ATP binding; phosphatase bindingBiological Process: nerve growth factor receptor signaling pathway; activation of MAPKK activity; viral reproduction; activation of MAPK activity; positive regulation of transcription, DNA-dependent; response to toxin; stress-activated MAPK cascade; toll-like receptor 3 signaling pathway; sensory perception of pain; protein amino acid phosphorylation; T cell receptor signaling pathway; toll-like receptor 5 signaling pathway; B cell receptor signaling pathway; regulation of transcription factor activity; small GTPase mediated signal transduction; toll-like receptor 4 signaling pathway; platelet activation; fibroblast growth factor receptor signaling pathway; transcription, DNA-dependent; toll-like receptor 2 signaling pathway; regulation of stress-activated MAPK cascade; nuclear translocation of MAPK; organ morphogenesis; response to estrogen stimulus; toll-like receptor 9 signaling pathway; regulation of cytoskeleton organization and biogenesis; axon guidance; positive regulation of translation; apoptosis; regulation of protein stability; chemotaxis; signal transduction; toll-like receptor 10 signaling pathway; synaptic transmission; response to exogenous dsRNA; positive regulation of cell proliferation; lipopolysaccharide-mediated signaling pathway; response to stress; epidermal growth factor receptor signaling pathway; MyD88-independent toll-like receptor signaling pathway; peptidyl-threonine phosphorylation; MAPKKK cascade; mammary gland epithelial cell proliferation; cell cycle; MyD88-dependent toll-like receptor signaling pathway; negative regulation of cell differentiation; peptidyl-serine phosphorylation; cytosine metabolic process; Ras protein signal transduction; insulin receptor signaling pathway; toll-like receptor signaling pathway; innate immune response; blood coagulation; vascular endothelial growth factor receptor signaling pathway; response to DNA damage stimulus; positive regulation of cell migration
UniProt Protein Details:
NCBI Summary:
This gene encodes a member of the MAP kinase family. MAP kinases, also known as extracellular signal-regulated kinases (ERKs), 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. The activation of this kinase requires its phosphorylation by upstream kinases. Upon activation, this kinase translocates to the nucleus of the stimulated cells, where it phosphorylates nuclear targets. One study also suggests that this protein acts as a transcriptional repressor independent of its kinase activity. The encoded protein has been identified as a moonlighting protein based on its ability to perform mechanistically distinct functions. Two alternatively spliced transcript variants encoding the same protein, but differing in the UTRs, have been reported for this gene. [provided by RefSeq, Jan 2014]
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.
