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Mouse Mixed lineage kinase domain-like protein (Mlkl) ELISA Kit (MOEB1658)

SKU:
MOEB1658
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q9D2Y4
ELISA Type:
Sandwich
Reactivity:
Mouse
€649
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Description

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Mouse Mixed lineage kinase domain-like protein (Mlkl) ELISA Kit

The Mouse Mixed Lineage Kinase Domain-Like Protein (MLKL) ELISA Kit is a highly sensitive and specific assay designed for the accurate detection of MLKL levels in mouse serum, plasma, and cell culture supernatants. This kit is ideal for researchers studying cell death pathways, necroptosis, and inflammation.MLKL is a key protein involved in necroptosis, a form of programmed cell death that is implicated in various inflammatory and autoimmune diseases. By accurately measuring MLKL levels, researchers can gain valuable insights into the mechanisms underlying these diseases and potentially identify new therapeutic targets.

With its high sensitivity and reproducibility, the Mouse MLKL ELISA Kit offers researchers a reliable tool for studying the role of MLKL in various physiological and pathological processes. Order now to accelerate your research and uncover new discoveries in the field of necroptosis and cell death pathways.

Product Name:Mouse Mixed lineage kinase domain-like protein (Mlkl) ELISA Kit
SKU:MOEB1658
Size:96T
Target:Mouse Mixed lineage kinase domain-like protein (Mlkl)
Synonyms:Mixed lineage kinase domain-like protein, Mlkl
Assay Type:Sandwich
Detection Method:ELISA
Reactivity:Mouse
Detection Range:0.312-20ng/mL
Sensitivity:0.18ng/mL
Intra CV:Provided with the Kit
Inter CV:Provided with the Kit
Linearity:Provided with the Kit
Recovery:Provided with the Kit
Function:Pseudokinase that plays a key role in TNF-induced necroptosis, a programmed cell death process. Activated following phosphorylation by RIPK3, leading to homotrimerization, localization to the plasma membrane and execution of programmed necrosis characterized by calcium influx and plasma membrane damage. Does not have protein kinase activity.
Uniprot:Q9D2Y4
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant mouse Mixed lineage kinase domain-like protein
Sub Unit:Homotrimer; forms homotrimers on necroptosis induction. Upon TNF-induced necrosis, forms in complex with PGAM5, RIPK1 and RIPK3. Within this complex, may play a role in the proper targeting of RIPK1/RIPK3 to its downstream effector PGAM5 (By similarity). Interacts with RIPK3; the interaction is direct.
Subcellular Location:Cytoplasm Cell membrane Localizes to the cytoplasm and translocates to the plasma membrane on necroptosis induction.
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:MLKL: Belongs to the protein kinase superfamily. 2 isoforms of the human protein are produced by alternative splicing.Protein type: Protein kinase, TKL; Protein kinase, Ser/Thr (non-receptor); Kinase, protein; TKL group; TKL-Unique familyCellular Component: membrane; cytoplasm; plasma membraneMolecular Function: transferase activity, transferring phosphorus-containing groups; protein complex binding; nucleotide binding; protein kinase binding; ATP binding; protein kinase activityBiological Process: programmed cell death; protein amino acid phosphorylation
UniProt Protein Details:
NCBI Summary:This gene belongs to the protein kinase superfamily. The encoded protein contains a protein kinase-like domain; however, is thought to lack protein kinase activity. This protein plays a critical role in tumor necrosis factor (TNF)-induced necroptosis, a programmed cell death process, via interaction with receptor-interacting protein 3 (Rip3), which is a key signaling molecule in necroptosis pathway. Knockout of this gene in mice showed that it is essential for necroptosis. Alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Sep 2015]
UniProt Code:Q9D2Y4
NCBI GenInfo Identifier:81916976
NCBI Gene ID:74568
NCBI Accession:Q9D2Y4.1
UniProt Secondary Accession:Q9D2Y4,Q8CIJ5
UniProt Related Accession:Q9D2Y4
Molecular Weight:54kDa
NCBI Full Name:Mixed lineage kinase domain-like protein
NCBI Synonym Full Names:mixed lineage kinase domain-like
NCBI Official Symbol:Mlkl
NCBI Official Synonym Symbols:9130019I15Rik
NCBI Protein Information:mixed lineage kinase domain-like protein; mlkl
UniProt Protein Name:Mixed lineage kinase domain-like protein
UniProt Synonym Protein Names:
Protein Family:Mixed lineage kinase domain-like protein
UniProt Gene Name:Mlkl
UniProt Entry Name:MLKL_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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