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Mouse Serine/threonine-protein kinase TBK1 (Tbk1) ELISA Kit (MOEB1136)

SKU:
MOEB1136
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q9WUN2
Range:
78-5000 pg/mL
ELISA Type:
Sandwich
Synonyms:
Tbk1, T2K, TANK-binding kinase 1
Reactivity:
Mouse
€649
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Description

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Mouse Serine/threonine-protein kinase TBK1 (Tbk1) ELISA Kit

The Mouse Serine/Threonine Protein Kinase TBK1 ELISA Kit is specifically designed for the accurate detection of TBK1 levels in mouse serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring precise and reproducible results for a variety of research applications.TBK1 is a key protein kinase involved in various cellular processes, including immune response, inflammation, and autophagy.

Dysregulation of TBK1 has been implicated in autoimmune diseases, infectious diseases, and cancer, highlighting its importance as a potential therapeutic target and biomarker for disease studies.With the Mouse Serine/Threonine Protein Kinase TBK1 ELISA Kit, researchers can confidently measure TBK1 levels in biological samples, aiding in the understanding of its role in health and disease.

Product Name:Mouse Serine/threonine-protein kinase TBK1 (Tbk1) ELISA Kit
SKU:MOEB1136
Size:96T
Target:Mouse Serine/threonine-protein kinase TBK1 (Tbk1)
Synonyms:T2K, TANK-binding kinase 1
Assay Type:Sandwich
Detection Method:ELISA
Reactivity:Mouse
Detection Range:78-5000pg/mL
Sensitivity:39.7pg/mL
Intra CV:5.4%
Inter CV:10.8%
Linearity:
Sample1:21:41:81:16
Serum(N=5)107-117%81-92%107-117%81-89%
EDTA Plasma(N=5)105-117%88-100%82-94%82-91%
Heparin Plasma(N=5)107-118%100-111%104-113%90-102%
Recovery:
Sample TypeAverage(%)Recovery Range(%)
Serum10195-107
Plasma10397-109
Function:Serine/threonine kinase that plays an essential role in regulating inflammatory responses to foreign agents. Following activation of toll-like receptors by viral or bacterial components, associates with TRAF3 and TANK and phosphorylates interferon regulatory factors (IRFs) IRF3 and IRF7 as well as DDX3X. This activity allows subsequent homodimerization and nuclear translocation of the IRFs leading to transcriptional activation of pro-inflammatory and antiviral genes including IFNA and IFNB. In order to establish such an antiviral state, TBK1 form several different complexes whose composition depends on the type of cell and cellular stimuli. Thus, several scaffolding molecules including FADD, TRADD, MAVS, AZI2, TANK or TBKBP1/SINTBAD can be recruited to the TBK1-containing-complexes. Under particular conditions, functions as a NF-kappa-B effector by phosphorylating NF-kappa-B inhibitor alpha/NFKBIA, IKBKB or RELA to translocate NF-Kappa-B to the nucleus. Restricts bacterial proliferation by phosphorylating the autophagy receptor OPTN/Optineurin on 'Ser-177', thus enhancing LC3 binding affinity and antibacterial autophagy. Phosphorylates and activates AKT1. Seems to play a role in energy balance regulation by sustaining a state of chronic, low-grade inflammation in obesity, wich leads to a negative impact on insulin sensitivity.
Uniprot:Q9WUN2
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant mouse Serine/threonine-protein kinase TBK1
Sub Unit:Homodimer. Interacts with DDX3X, TIRAP and TRAF2. Part of a ternary complex consisting of TANK, TRAF2 and TBK1. Interacts with AZI2, TANK and TBKBP1; these interactions are mutually exclusive and mediate TBK1 activation. Interacts with GSK3B; this interaction promotes TBK1 self-association and autophosphorylation. Interacts with SIKE1; SIKE1 is associated with TBK1 under physiological condition and dissociated from TBK1 upon viral infection or TLR3 stimulation. Interacts with IRF3 and DDX58/RIG-I. Interacts with CYLD. Interacts with OPTN and TRAF3. Interacts with SRC. Interacts with the exocyst complex subunit SEC5/EXOC2; this interaction is sufficient to trigger TBK1 activity. Interacts with TMEM173/MITA. Interacts with IFIT3 (via N-terminus). Interacts with MAVS only in the presence of IFIT3. Interacts with TICAM1 and this interaction is enhanced in the presence of WDFY1.
Research Area:Immunology
Subcellular Location:Cytoplasm Upon mitogen stimulation or triggering of the immune system, TBK1 is recruited to the exocyst by EXOC2.
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:TBK1: a protein kinase of the IKK family. Can mediate NFkB activation in response to certain growth factors. Forms a complex with the IKB protein TANK and TRAF2 and release the NFKB inhibition caused by TANK.
UniProt Protein Details:

Protein type:Protein kinase, Ser/Thr (non-receptor); EC 2.7.11.1; Protein kinase, Other; Kinase, protein; Other group; IKK family

Cellular Component: cytoplasm

Molecular Function:nucleic acid binding; phosphoprotein binding; protein binding; protein serine/threonine kinase activity

Biological Process: activation of innate immune response; defense response to Gram-positive bacterium; peptidyl-serine phosphorylation; positive regulation of interferon-alpha production; positive regulation of interferon-beta biosynthetic process; positive regulation of interferon-beta production; positive regulation of transcription from RNA polymerase II promoter

UniProt Code:Q9WUN2
NCBI GenInfo Identifier:81917888
NCBI Gene ID:56480
NCBI Accession:Q9WUN2.1
UniProt Secondary Accession:Q9WUN2,Q9CT90, Q9DC03,
UniProt Related Accession:Q9WUN2
Molecular Weight:83,425 Da
NCBI Full Name:Serine/threonine-protein kinase TBK1
NCBI Synonym Full Names:TANK-binding kinase 1
NCBI Official Symbol:Tbk1
NCBI Official Synonym Symbols:AI462036; AW048562; 1200008B05Rik
NCBI Protein Information:serine/threonine-protein kinase TBK1
UniProt Protein Name:Serine/threonine-protein kinase TBK1
UniProt Synonym Protein Names:T2K; TANK-binding kinase 1
Protein Family:Serine/threonine-protein kinase
UniProt Gene Name:Tbk1
UniProt Entry Name:TBK1_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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