The Mouse Serine/Threonine Protein Kinase STK11 (STK11) ELISA Kit is specifically designed for the accurate measurement of STK11 levels in mouse serum, plasma, and cell culture supernatants. This kit is highly sensitive and specific, ensuring reliable and reproducible results for a variety of research applications.STK11 is a key enzyme involved in regulating cell growth and metabolism, playing a critical role in various biological processes.
Dysregulation of STK11 has been implicated in a variety of diseases, including cancer, metabolic disorders, and inflammatory conditions. Therefore, this ELISA kit serves as a valuable tool for studying the role of STK11 in disease pathogenesis and therapeutic development.
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse Serine/threonine-protein kinase STK11
Sub Unit:
Catalytic component of a trimeric complex composed of STK11/LKB1, STRAD (STRADA or STRADB) and CAB39/MO25 (CAB39/MO25alpha or CAB39L/MO25beta): the complex tethers STK11/LKB1 in the cytoplasm and stimulates its catalytic activity. Found in a ternary complex composed of SMAD4, STK11/LKB1 and STK11IP. Interacts with p53/TP53, SMAD4, STK11IP and WDR6. Interacts with NR4A1 (By similarity). Interacts with NISCH; this interaction may increase STK11 activity (By similarity). Interacts with PTEN, leading to PTEN phosphorylation (By similarity). Interacts with SIRT1; the interaction deacetylates STK11 (By similarity). Interacts with CDKN1A.
Research Area:
Cancer
Subcellular Location:
Isoform 2 Nucleus Cytoplasm Relocates to the cytoplasm when bound to STRAD (STRADA or STRADB) and CAB39/MO25 (CAB39/MO25alpha or CAB39L/MO25beta).
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]
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.
