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Mouse Mitogen-activated protein kinase kinase kinase 5 (Map3k5) ELISA Kit (MOEB1206)

SKU:
MOEB1206
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
O35099
ELISA Type:
Sandwich
Synonyms:
ASK1, MAP3K5, MEKK5
Reactivity:
Mouse
€649
Frequently bought together:

Description

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Mouse Mitogen-activated protein kinase kinase kinase 5 (Map3k5) ELISA Kit

The Mouse Mitogen-Activated Protein Kinase Kinase Kinase 5 (MAP3K5) ELISA Kit is specifically designed for the accurate detection of MAP3K5 levels in mouse samples, including serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring reliable and reproducible results for a variety of research applications.MAP3K5 is a key signaling protein involved in cell growth, development, and immune response. It plays a crucial role in various biological processes, including inflammation, apoptosis, and cell differentiation.

Dysregulation of MAP3K5 activity has been linked to a variety of diseases, including cancer, autoimmune disorders, and neurodegenerative diseases, making it an important biomarker for understanding these conditions and developing potential therapies.Overall, the Mouse MAP3K5 ELISA Kit provides researchers with a valuable tool for studying the function and regulation of MAP3K5 in mouse models, ultimately advancing our understanding of cellular signaling pathways and disease mechanisms.

Product Name:Mouse Mitogen-activated protein kinase kinase kinase 5 (Map3k5) ELISA Kit
SKU:MOEB1206
Size:96T
Target:Mouse Mitogen-activated protein kinase kinase kinase 5 (Map3k5)
Synonyms:Apoptosis signal-regulating kinase 1, MAPK/ERK kinase kinase 5, ASK-1, MEK kinase 5, Ask1, Mekk5
Assay Type:Sandwich
Detection Method:ELISA
Reactivity:Mouse
Detection Range:0.312-20ng/mL
Sensitivity:0.156ng/mL
Intra CV:Provided with the Kit
Inter CV:Provided with the Kit
Linearity:Provided with the Kit
Recovery:Provided with the Kit
Function:Serine/threonine kinase which acts as an essential component of the MAP kinase signal transduction pathway. Plays an important role in the cascades of cellular responses evoked by changes in the environment. Mediates signaling for determination of cell fate such as differentiation and survival. Plays a crucial role in the apoptosis signal transduction pathway through mitochondria-dependent caspase activation. MAP3K5/ASK1 is required for the innate immune response, which is essential for host defense against a wide range of pathogens. Mediates signal transduction of various stressors like oxidative stress as well as by receptor-mediated inflammatory signals, such as the tumor necrosis factor (TNF) or lipopolysaccharide (LPS). Once activated, acts as an upstream activator of the MKK/JNK signal transduction cascade and the p38 MAPK signal transduction cascade through the phosphorylation and activation of several MAP kinase kinases like MAP2K4/SEK1, MAP2K3/MKK3, MAP2K6/MKK6 and MAP2K7/MKK7. These MAP2Ks in turn activate p38 MAPKs and c-jun N-terminal kinases (JNKs). Both p38 MAPK and JNKs control the transcription factors activator protein-1 (AP-1).
Uniprot:O35099
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant mouse Mitogen-activated protein kinase kinase kinase 5
Sub Unit:Homodimer when inactive (By similarity). Binds both upstream activators and downstream substrates in multimolecular complexes. Part of a cytoplasmic complex made of HIPK1, DAB2IP and MAP3K5 in response to TNF (By similarity). This complex formation promotes MAP3K5-JNK activation and subsequent apoptosis (By similarity). Interacts with SOCS1 which recognizes phosphorylation of Tyr-725 and induces MAP3K5/ASK1 degradation in endothelial cells (By similarity). Interacts with the 14-3-3 family proteins such as YWHAB, YWHAE, YWHAQ, YWHAH, YWHAZ and SFN (By similarity). Interacts with ARRB2, BIRC2, DAB2IP, IGF1R, MAP3K6/ASK2, PIM1, PGAM5, PPP5C, SOCS1, STUB1, TRAF2 and TXN (By similarity). Interacts with ERN1 in a TRAF2-dependent manner (By similarity). Interacts with calcineurin subunit PPP3R1, PPM1L and TRAF6. Interacts (via N-terminus) with RAF1 and this interaction inhibits the proapoptotic function of MAP3K5. Interacts with DAB2IP (via N-terminus C2 domain); the interaction occurs in a TNF-alpha-dependent manner (By similarity).Interacts with DUSP13/DUSP13A; may positively regulate apoptosis.
Research Area:Neurosciences
Subcellular Location:Cytoplasm Endoplasmic reticulum Interaction with 14-3-3 proteins alters the distribution of MAP3K5/ASK1 and restricts it to the perinuclear endoplasmic reticulum region.
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:ASK1: a MAP kinase kinase kinase of the STE11 family, apoptosis signal-regulating kinase 1 (ASK1) plays essential roles in stress-induced apoptosis. Activated by various stressors, including oxidative stress, endoplasmic reticulum stress, and calcium overload, as well as by receptor-mediated inflammatory signals, such as the tumor necrosis factor (TNF) and lipopolysaccharide (LPS). In turn, ASK1 phosphorylates and activates two different subgroups of MAP kinase kinases, MKK4/SEK1 and MKK3/MKK6, which in turn activate stress-activated protein kinases (JNKs) and p38 subgroups of MAP kinases, respectively. Overexpression activates JNK and p38 and induces apoptosis in several cell types through signals involving the mitochondrial cell death pathway. Embryonic fibroblasts or primary neurons derived from ASK1-/- mice are resistant to stress-induced JNK and p38 activation as well as cell death. Oxidative stress induces the activation of ASK1, which correlate with ASK1 activity and ASK1-dependent apoptosis. Required for the innate immune response, which is essential for host defense against a wide range of pathogens.
UniProt Protein Details:

Protein type:EC 2.7.11.25; Protein kinase, Ser/Thr (non-receptor); Kinase, protein; Protein kinase, STE; STE group; STE11 family

Cellular Component: cytoplasm; cytosol; external side of plasma membrane

Molecular Function:ATP binding; magnesium ion binding; MAP kinase kinase kinase activity; protein binding; protein homodimerization activity; protein kinase activity; protein kinase binding; protein phosphatase binding

Biological Process: activation of MAPK activity; activation of MAPKK activity; induction of apoptosis by oxidative stress; JNK cascade; MAPKKK cascade; positive regulation of apoptosis; positive regulation of caspase activity; positive regulation of JNK activity; positive regulation of JNK cascade; positive regulation of myoblast differentiation; positive regulation of protein amino acid phosphorylation; protein amino acid phosphorylation

UniProt Code:O35099
NCBI GenInfo Identifier:341941007
NCBI Gene ID:26408
NCBI Accession:O35099.3
UniProt Secondary Accession:O35099,Q14AY5,
UniProt Related Accession:O35099
Molecular Weight:154,512 Da
NCBI Full Name:Mitogen-activated protein kinase kinase kinase 5
NCBI Synonym Full Names:mitogen-activated protein kinase kinase kinase 5
NCBI Official Symbol:Map3k5
NCBI Official Synonym Symbols:ASK; ASK1; Mekk5; MAPKKK5; 7420452D20Rik
NCBI Protein Information:mitogen-activated protein kinase kinase kinase 5
UniProt Protein Name:Mitogen-activated protein kinase kinase kinase 5
UniProt Synonym Protein Names:Apoptosis signal-regulating kinase 1; ASK-1; MAPK/ERK kinase kinase 5; MEK kinase 5; MEKK 5
Protein Family:Mitogen-activated protein kinase kinase kinase
UniProt Gene Name:Map3k5
UniProt Entry Name:M3K5_MOUSE
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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