Human IKBKE ELISA Kit (HUEB1635)- High Sensitivity

Product Name:	Human Inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKBKE) ELISA Kit
SKU:	HUEB1635
Size:	96T
Target:	Human Inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKBKE)
Synonyms:	Inducible I kappa-B kinase, IKK-i, I-kappa-B kinase epsilon, IKKE, IKKI, KIAA0151
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	78-5000pg/mL
Sensitivity:	49pg/ml

Intra CV:

8.4%

Inter CV:

11.1%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	109-119%	103-114%	104-114%	97-105%
EDTA Plasma(N=5)	99-108%	90-99%	93-102%	99-108%
Heparin Plasma(N=5)	102-110%	109-119%	98-108%	96-107%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	107	101-113
Plasma	109	103-115

Function:

Serine/threonine kinase that plays an essential role in regulating inflammatory responses to viral infection, through the activation of the type I IFN, NF-kappa-B and STAT signaling. Also involved in TNFA and inflammatory cytokines, like Interleukin-1, signaling. Following activation of viral RNA sensors, such as RIG-I-like receptors, associates with DDX3X and phosphorylates interferon regulatory factors (IRFs), IRF3 and IRF7, as well as DDX3X. This activity allows subsequent homodimerization and nuclear translocation of the IRF3 leading to transcriptional activation of pro-inflammatory and antiviral genes including IFNB. In order to establish such an antiviral state, IKBKE forms several different complexes whose composition depends on the type of cell and cellular stimuli. Thus, several scaffolding molecules including IPS1/MAVS, TANK, AZI2/NAP1 or TBKBP1/SINTBAD can be recruited to the IKBKE-containing-complexes. Activated by polyubiquitination in response to TNFA and interleukin-1, regulates the NF-kappa-B signaling pathway through, at least, the phosphorylation of CYLD. Phosphorylates inhibitors of NF-kappa-B thus leading to the dissociation of the inhibitor/NF-kappa-B complex and ultimately the degradation of the inhibitor. In addition, is also required for the induction of a subset of ISGs which displays antiviral activity, may be through the phosphorylation of STAT1 at 'Ser-708'. Phosphorylation of STAT1 at 'Ser-708' seems also to promote the assembly and DNA binding of ISGF3 (STAT1:STAT2:IRF9) complexes compared to GAF (STAT1:STAT1) complexes, in this way regulating the balance between type I and type II IFN responses. Protects cells against DNA damage-induced cell death. Also plays an important 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. Phosphorylates AKT1.

Uniprot:	Q14164
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Inhibitor of nuclear factor kappa-B kinase subunit epsilon
Sub Unit:	Homodimer. Interacts with MAVS/IPS1. Interacts with the adapter proteins AZI2/NAP1, TANK and TBKBP1/SINTBAD. Interacts with SIKE1. Interacts with TICAM1/TRIF, IRF3 and DDX58/RIG-I; interactions are disrupted by the interaction between IKBKE and SIKE1. Interacts with TOPORS; induced by DNA damage. Interacts with CYLD. Interacts (when polyubiquitinated) with IKBKB, IKBKG and MYD88. Interacts with IFIH1 (PubMed:17600090). Interacts with DDX3X; the interaction is found to be induced upon virus infection. Interacts (via Protein kinase domain) with arenavirus protein N; the interaction inhibits IKBKE kinase function. Interacts with Ebola virus protein VP35; the interaction leads to inhibition of cellular antiviral response by blocking necessary interactions between the IKBKE and MAVS/IPS as well as its substrates IRF3 and IRF7. Interacts with TRIM6 (via SPRY box) (PubMed:24882218). Interacts with unanchored K48-linked polyubiquitin chains; this leads to IKBKE activation (PubMed:24882218).
Research Area:	Cancer
Subcellular Location:	Cytoplasm Nucleus Nucleus PML body Targeting to PML nuclear bodies upon DNA damage is TOPORS-dependent (PubMed:20188669). Located diffusely throughout the cytoplasm but locates to punctate cytoplasmic bodies when coexpressed with TRIM6 (PubMed:24882218).
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	IKKE: a oncogenic kinase of the IKK family. Phosphorylates inhibitors of NF-kappa-B thus leading to the dissociation of the inhibitor/NF-kappa-B complex and ultimately the degradation of the inhibitor. Plays a pivotal role in coordinating the activation of IRF3 and NF-kappaB in the innate immune response. Its redistribution to PML nuclear bodies upon DNA damage is TOPORS-dependent. Is a breast cancer oncogene that exerts its oncogenic properties through cRel.
UniProt Protein Details:	Protein type:Inhibitor; Kinase, protein; EC 2.7.11.10; Protein kinase, Other; Protein kinase, Ser/Thr (non-receptor); Other group; IKK family Chromosomal Location of Human Ortholog: 1q32.1 Cellular Component: nucleoplasm; PML body; cytoplasm; mitochondrial membrane; endosome membrane; nucleus; cytosol Molecular Function:NF-kappaB-inducing kinase activity; protein binding; IkappaB kinase activity; ATP binding Biological Process: positive regulation of I-kappaB kinase/NF-kappaB cascade; DNA damage response, signal transduction resulting in induction of apoptosis; MyD88-independent toll-like receptor signaling pathway; toll-like receptor signaling pathway; innate immune response; immune response; toll-like receptor 3 signaling pathway; I-kappaB phosphorylation; negative regulation of interferon type I production; toll-like receptor 4 signaling pathway; protein amino acid phosphorylation
NCBI Summary:	IKBKE is a noncanonical I-kappa-B (see MIM 164008) kinase (IKK) that is essential for regulating antiviral signaling pathways. IKBKE has also been identified as a breast cancer (MIM 114480) oncogene and is amplified and overexpressed in over 30% of breast carcinomas and breast cancer cell lines (Hutti et al., 2009 [PubMed 19481526]).[supplied by OMIM, Oct 2009]
UniProt Code:	Q14164
NCBI GenInfo Identifier:	14548079
NCBI Gene ID:	9641
NCBI Accession:	Q14164.1
UniProt Secondary Accession:	Q14164,Q3B754, Q3KR43, Q5JTS6, D3DT78,
UniProt Related Accession:	Q14164
Molecular Weight:	716
NCBI Full Name:	Inhibitor of nuclear factor kappa-B kinase subunit epsilon
NCBI Synonym Full Names:	inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon
NCBI Official Symbol:	IKBKE
NCBI Official Synonym Symbols:	IKKE; IKKI; IKK-E; IKK-i
NCBI Protein Information:	inhibitor of nuclear factor kappa-B kinase subunit epsilon; IKK-epsilon; I-kappa-B kinase epsilon; inducible IkappaB kinase; IKK-related kinase epsilon; inducible I kappa-B kinase
UniProt Protein Name:	Inhibitor of nuclear factor kappa-B kinase subunit epsilon
UniProt Synonym Protein Names:	Inducible I kappa-B kinase; IKK-i
UniProt Gene Name:	IKBKE
UniProt Entry Name:	IKKE_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

Human IKBKE / Inhibitor of nuclear factor kappa-B kinase subunit epsilon ELISA Kit