Mouse Serine/threonine-protein kinase STK11 (Stk11) ELISA Kit

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SKU:
MOEB1139
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q9WTK7
ELISA Type:
Sandwich
Reactivity:
Mouse
Frequently bought together:

Description

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Product Name:Mouse Serine/threonine-protein kinase STK11 (Stk11) ELISA Kit
Product Code:MOEB1139
Alias:Serine/threonine-protein kinase STK11, Liver kinase B1 homolog, LKB1, mLKB1, Stk11, Lkb1, 2.7.11.1
Uniprot:Q9WTK7
Reactivity:Mouse
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:LKB1: a S/T protein kinase of the CAMKL family. A tumor suppressor that helps control cell structure, polarity, apoptosis and energy homeostasis. Phosphorylates AGS3 (activator of G-protein signaling 3) GPR domains, regulating the interaction of GPR-containing proteins with G-proteins. A cytosolic protein complex comprised of LKB1, the pseudokinase STRAD, and the MO25 scaffold protein. Activates AMPK and several related protein kinases. AMPK plays a predominant role as the master regulator of cellular energy homeostasis, controlling downstream effectors that regulate cell growth and apoptosis in response to cellular ATP concentrations. The Lkb1/Strad/Mo25 complex can polarize single epithelial cells, leading to the formation of a brush border containing microvilli on the apical surface. Mutation in the corresponding gene causes Peutz-Jeghers syndrome (PJS), an autosomal dominant disorder characterized by benign GI tract polyps and dark skin lesions of the mouth, hands and feet. A variety of other LKB1 gene mutations have been associated with the formation of sporadic cancers in several tissues.Protein type: Protein kinase, CAMK; Kinase, protein; Tumor suppressor; EC 2.7.11.1; Protein kinase, Ser/Thr (non-receptor); Autophagy; CAMK group; CAMKL family; LKB subfamilyCellular Component: cytoplasm; cytosol; membrane; mitochondrion; nucleus; protein complexMolecular Function: ATP binding; LRR domain binding; magnesium ion binding; p53 binding; protein binding; protein complex binding; protein kinase activator activity; protein serine/threonine kinase activityBiological Process: activation of protein kinase activity; anoikis; axonogenesis; cell cycle arrest; establishment of cell polarity; glucose homeostasis; Golgi localization; negative regulation of cell growth; negative regulation of cell proliferation; negative regulation of lipid biosynthetic process; positive regulation of autophagy; positive regulation of axonogenesis; positive regulation of gluconeogenesis; positive regulation of peptidyl-tyrosine phosphorylation; positive regulation of transforming growth factor beta receptor signaling pathway; positive thymic T cell selection; protein amino acid autophosphorylation; protein amino acid phosphorylation; protein heterooligomerization; regulation of cell growth; regulation of dendrite morphogenesis; regulation of protein kinase B signaling cascade; regulation of Wnt receptor signaling pathway; response to DNA damage stimulus; response to ionizing radiation; response to lipid; spermatid development; T cell receptor signaling pathway; tissue homeostasis; vasculature development; Wnt receptor signaling pathway through beta-catenin
UniProt Protein Details:
NCBI Summary:This gene encodes a member of the serine/threonine kinase family. The encoded protein, a known tumor suppressor, activates (via phosphorylation) adenine monophosphate-activated protein kinase (AMPK) and AMPK-related kinase proteins. This upstream regulation of the AMPK pathway is thought to regulate a number of different processes, including cell metabolism, cell polarity, apoptosis and DNA damage response. Mutations in a similar gene in human have been associated with Peutz-Jeghers syndrome. Alternative splicing results in multiple transcript variants, including the S isoform which plays a potential role in spermiogenesis. [provided by RefSeq, Sep 2014]
UniProt Code:Q9WTK7
NCBI GenInfo Identifier:81917862
NCBI Gene ID:20869
NCBI Accession:Q9WTK7.1
UniProt Secondary Accession:Q9WTK7,Q3TAE0, B3VBP0
UniProt Related Accession:Q9WTK7
Molecular Weight:46,494 Da
NCBI Full Name:Serine/threonine-protein kinase STK11
NCBI Synonym Full Names:serine/threonine kinase 11
NCBI Official Symbol:Stk11  
NCBI Official Synonym Symbols:Lkb1; Par-4; R75140; AA408040  
NCBI Protein Information:serine/threonine-protein kinase STK11
UniProt Protein Name:Serine/threonine-protein kinase STK11
UniProt Synonym Protein Names:Liver kinase B1 homolog; LKB1; mLKB1
Protein Family:Serine/threonine-protein kinase
UniProt Gene Name:Stk11  
UniProt Entry Name:STK11_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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