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Rat MAP kinase-activated protein kinase 3 (Mapkapk3) ELISA Kit (RTEB1136)

SKU:
RTEB1136
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q66H84
Range:
0.312-20 ng/mL
ELISA Type:
Sandwich
Reactivity:
Rat
$779
Frequently bought together:

Description

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Rat MAP kinase-activated protein kinase 3 (Mapkapk3) ELISA Kit

The Rat MAP Kinase-Activated Protein Kinase 3 (MAPKAPK3) ELISA Kit is a powerful tool for detecting and quantifying levels of MAPKAPK3 in rat samples. This kit offers high sensitivity and specificity, providing accurate and reliable results for various research applications.MAPKAPK3 is a key player in the MAPK signaling pathway, regulating cellular responses to external stimuli and stress. Its dysregulation has been implicated in various diseases, including cancer, inflammation, and neurodegenerative disorders.

Studying MAPKAPK3 levels can provide valuable insights into disease mechanisms and potential therapeutic targets.With this ELISA kit, researchers can easily measure MAPKAPK3 levels in rat serum, plasma, or cell culture supernatants, facilitating in-depth analysis of the protein's function and its role in disease progression. Stay ahead in your research with the Rat MAPKAPK3 ELISA Kit from Assay Genie.

Product Name:Rat MAP kinase-activated protein kinase 3 (Mapkapk3) ELISA Kit
SKU:RTEB1136
Size:96T
Target:Rat MAP kinase-activated protein kinase 3 (Mapkapk3)
Synonyms:MAPK-activated protein kinase 3
Assay Type:Sandwich
Detection Method:ELISA
Reactivity:Rat
Detection Range:0.312-20ng/mL
Sensitivity:0.186ng/mL
Intra CV:3.8%
Inter CV:6.9%
Linearity:
Sample1:21:41:81:16
Serum(N=5)82-92%94-103%105-115%85-97%
EDTA Plasma(N=5)103-113%93-106%101-111%105-116%
Heparin Plasma(N=5)80-90%110-119%88-99%96-106%
Recovery:
Sample TypeAverage(%)Recovery Range(%)
Serum8180-87
Plasma8380-89
Function:Stress-activated serine/threonine-protein kinase involved in cytokines production, endocytosis, cell migration, chromatin remodeling and transcriptional regulation. Following stress, it is phosphorylated and activated by MAP kinase p38-alpha/MAPK14, leading to phosphorylation of substrates. Phosphorylates serine in the peptide sequence, Hyd-X-R-X(2)-S, where Hyd is a large hydrophobic residue. MAPKAPK2 and MAPKAPK3, share the same function and substrate specificity, but MAPKAPK3 kinase activity and level in protein expression are lower compared to MAPKAPK2. Phosphorylates HSP27/HSPB1, KRT18, KRT20, RCSD1, RPS6KA3, TAB3 and TTP/ZFP36. Mediates phosphorylation of HSP27/HSPB1 in response to stress, leading to dissociate HSP27/HSPB1 from large small heat-shock protein (sHsps) oligomers and impair their chaperone activities and ability to protect against oxidative stress effectively. Involved in inflammatory response by regulating tumor necrosis factor (TNF) and IL6 production post-transcriptionally: acts by phosphorylating AU-rich elements (AREs)-binding proteins, such as TTP/ZFP36, leading to regulate the stability and translation of TNF and IL6 mRNAs. Phosphorylation of TTP/ZFP36, a major post-transcriptional regulator of TNF, promotes its binding to 14-3-3 proteins and reduces its ARE mRNA affinity leading to inhibition of dependent degradation of ARE-containing transcript. Involved in toll-like receptor signaling pathway (TLR) in dendritic cells: required for acute TLR-induced macropinocytosis by phosphorylating and activating RPS6KA3. Also acts as a modulator of Polycomb-mediated repression.
Uniprot:Q66H84
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant rat MAP kinase-activated protein kinase 3
Sub Unit:Heterodimer with p38-alpha/MAPK14. The heterodimer with p38-alpha/MAPK14 forms a stable complex: molecules are positioned 'face to face' so that the ATP-binding sites of both kinases are at the heterodimer interface. Interacts with TCF3 and with polycomb proteins, such as PCH2 and BMI1/PCGF4.
Research Area:Cancer
Subcellular Location:Nucleus Cytoplasm Predominantly located in the nucleus, when activated it translocates to the cytoplasm.
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:MAPKAPK3: a member of the MAPKAPK family of protein kinases. Activated by growth inducers and stress stimulation of cells. In vitro studies demonstrated that ERK, p38 MAP kinase and Jun N-terminal kinase were all able to phosphorylate and activate this kinase, which suggested the role of this kinase as an integrative element of signaling in both mitogen and stress responses. This kinase was reported to interact with, phosphorylate and repress the activity of E47, which is a basic helix-loop-helix transcription factor known to be involved in the regulation of tissue-specific gene expression and cell differentiation.Protein type: Protein kinase, Ser/Thr (non-receptor); Kinase, protein; EC 2.7.11.1; Protein kinase, CAMK; CAMK group; MAPKAPK family; MAPKAPK subfamilyCellular Component: cytoplasm; nuclear membrane; nucleoplasmMolecular Function: ATP binding; calcium-dependent protein serine/threonine kinase activity; calmodulin binding; calmodulin-dependent protein kinase activity; protein serine/threonine kinase activity; signal transducer activityBiological Process: cell surface receptor linked signal transduction; MAPKKK cascade; peptidyl-serine phosphorylation; protein amino acid autophosphorylation; response to cytokine stimulus; response to lipopolysaccharide; toll-like receptor signaling pathway
UniProt Protein Details:
NCBI Summary:
UniProt Code:Q66H84
NCBI GenInfo Identifier:58865824
NCBI Gene ID:315994
NCBI Accession:NP_001012127.1
UniProt Secondary Accession:Q66H84
UniProt Related Accession:Q66H84
Molecular Weight:43,222 Da
NCBI Full Name:MAP kinase-activated protein kinase 3
NCBI Synonym Full Names:mitogen-activated protein kinase-activated protein kinase 3
NCBI Official Symbol:Mapkapk3
NCBI Official Synonym Symbols:MK-3; MAPKAP-K3
NCBI Protein Information:MAP kinase-activated protein kinase 3
UniProt Protein Name:MAP kinase-activated protein kinase 3
UniProt Synonym Protein Names:
Protein Family:MAP kinase-activated protein kinase
UniProt Gene Name:Mapkapk3
UniProt Entry Name:MAPK3_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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