Human TBK1 ELISA Kit (HUEB1371)- High Sensitivity

Product Name:	Human Serine/threonine-protein kinase TBK1 (TBK1) ELISA Kit
SKU:	HUEB1371
Size:	96T
Target:	Human Serine/threonine-protein kinase TBK1 (TBK1)
Synonyms:	NF-kappa-B-activating kinase, T2K, TANK-binding kinase 1, NAK
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.156-10ng/mL
Sensitivity:	0.078ng/mL

Intra CV:

8.1%

Inter CV:

10.2%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	86-96%	99-109%	84-97%	100-111%
EDTA Plasma(N=5)	112-121%	98-108%	91-101%	96-107%
Heparin Plasma(N=5)	81-91%	102-112%	101-110%	107-117%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	94	88-100
Plasma	96	90-102

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. Attenuates retroviral budding by phosphorylating the endosomal sorting complex required for transport-I (ESCRT-I) subunit VPS37C. Phosphorylates Borna disease virus (BDV) P protein.

Uniprot:	Q9UHD2
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Serine/threonine-protein kinase TBK1
Sub Unit:	(Microbial infection) Interacts with HCV NS3; this interaction leads to inhibition of cellular antiviral response by blocking necessary interactions between the TBK1 and its substrates IRF3 and IRF7.
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, Other; Protein kinase, Ser/Thr (non-receptor); Kinase, protein; EC 2.7.11.1; Other group; IKK family Chromosomal Location of Human Ortholog: 12q14.1 Cellular Component: cytoplasm; endosome membrane; cytosol Molecular Function:protein serine/threonine kinase activity; protein binding; nucleic acid binding; phosphoprotein binding; ATP binding; protein kinase activity Biological Process: I-kappaB kinase/NF-kappaB cascade; positive regulation of I-kappaB kinase/NF-kappaB cascade; viral reproduction; MyD88-independent toll-like receptor signaling pathway; response to virus; toll-like receptor 3 signaling pathway; protein amino acid phosphorylation; positive regulation of peptidyl-serine phosphorylation; defense response to Gram-positive bacterium; positive regulation of interferon-beta biosynthetic process; positive regulation of interferon type I production; positive regulation of interferon-beta production; toll-like receptor signaling pathway; interferon type I production; innate immune response; positive regulation of transcription from RNA polymerase II promoter; inflammatory response; negative regulation of interferon type I production; toll-like receptor 4 signaling pathway; defense response to virus; positive regulation of interferon-alpha production Disease: Frontotemporal Dementia And/or Amyotrophic Lateral Sclerosis 4
NCBI Summary:	The NF-kappa-B (NFKB) complex of proteins is inhibited by I-kappa-B (IKB) proteins, which inactivate NFKB by trapping it in the cytoplasm. Phosphorylation of serine residues on the IKB proteins by IKB kinases marks them for destruction via the ubiquitination pathway, thereby allowing activation and nuclear translocation of the NFKB complex. The protein encoded by this gene is similar to IKB kinases and can mediate NFKB activation in response to certain growth factors. [provided by RefSeq, Oct 2010]
UniProt Code:	Q9UHD2
NCBI GenInfo Identifier:	74761953
NCBI Gene ID:	29110
NCBI Accession:	Q9UHD2.1
UniProt Secondary Accession:	Q9UHD2,Q8IYV3, Q9NUJ5, A8K4S4,
UniProt Related Accession:	Q9UHD2
Molecular Weight:	83,642 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:	NAK; T2K
NCBI Protein Information:	serine/threonine-protein kinase TBK1; NF-kB-activating kinase; NF-kappa-B-activating kinase
UniProt Protein Name:	Serine/threonine-protein kinase TBK1
UniProt Synonym Protein Names:	NF-kappa-B-activating kinase; T2K; TANK-binding kinase 1
Protein Family:	Serine/threonine-protein kinase
UniProt Gene Name:	TBK1
UniProt Entry Name:	TBK1_HUMAN

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.

ELISA	Antibodies
Human TBK1 (Serine/threonine-protein kinase TBK1) ELISA Kit (HUFI04825)	Anti-TBK1 Antibody (CAB2573)
	Anti-NAK/TBK1 (N-term) Antibody (CAB3458)
	Anti-Phospho-TBK1-S172 pAb Antibody (CABP0847)
	Anti-Phospho-NAK/TBK1-S172 Antibody (CABP1026)
	TBK1 Antibody (PACO03991)