Human KEAP1 ELISA Kit (HUFI03108)- High Sensitivity

Product Name:	Human KEAP1 (kelch-like ECH-associated protein 1) ELISA Kit
Product Code:	HUFI03108
Size:	96 Assays
Alias:	INrf2, KIAA0132, KLHL19, MGC10630, MGC1114, MGC20887, MGC4407, MGC9454, cytosolic inhibitor of Nrf2
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human KEAP1 concentrations in serum plasma and other biological fluids.
Sensitivity:	14.063pg/ml
Range:	23.438-1500pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:	Matrices listed below were spiked with certain level of Human KEAP1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human KEAP1 in samples. Enquire for more information.
Linearity:	The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human KEAP1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Enquire for more information.
CV(%):	Intra-Assay: CV<8% Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	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

Uniprot

Q14145

UniProt Protein Function:	KEAP1: a substrate adapter protein for the E3 ubiquitin ligase complex formed by CUL3 and RBX1. Has tumor suppressor activity. Retains NRF2, BPTF and PGAM5 in the cytosol. Targets NRF2 for ubiquitination and degradation by the proteasome, thus resulting in the suppression of its transcriptional activity and of antioxidant response element-mediated detoxifying enzyme gene expression. Somatic mutations of KEAP1 or NRF2 commonly occur in solid cancers, irrespective of histological type, resulting in the constitutive transcription of cytoprotective genes. Genetic disruption of the KEAP1/CUL3 E3 ubiquitin ligase complex leads to the activation of the NF-kappaB pathway in lung cancer. Alternative activation of the cytoprotective NRF2 pathway exist in the absence of mutations in KEAP1 and HRF2: WTX, PALB2, SQSTM1, and DPP3 can competitively bind KEAP1. These proteins and others that bind KEAP1 contain an ÂƒÂƒÂƒÂƒÂ†Â’Â€ÂšÂ¿ETGEÂƒÂƒÂƒÂƒÂ†Â’Â€ÂšÂ¿ amino acid motif, which matches the KEAP1 interaction motif found in NRF2. Binding of these ÂƒÂƒÂƒÂƒÂ†Â’Â€ÂšÂ¿ETGEÂƒÂƒÂƒÂƒÂ†Â’Â€ÂšÂ¿-containing proteins can displace NRF2, freeing it to translocate to the nucleus and implement its cytoprotective transcriptional pathway. The DPP3 gene is frequently overexpressed in squamous cell lung carcinomas with high wt NRF2 activity. Its expression level significantly correlates with the response rate and progression-free survival of platinum-based first-line chemotherapy. May be a useful biomarker for predicting the chemosensitivity of patients with advanced-stage NSCLC. Broadly expressed, with highest levels in skeletal muscle. Ubiquitination and subsequent degradation of PGAM5 is inhibited by oxidative stress and sulforaphane.
UniProt Protein Details:	Protein type:Ubiquitin conjugating system; Transcription regulation; Motility/polarity/chemotaxis; Endoplasmic reticulum Chromosomal Location of Human Ortholog: 19p13.2 Cellular Component: nucleoplasm; endoplasmic reticulum; cytoplasm; microtubule organizing center; midbody; cytosol Molecular Function:protein binding; transcription factor binding Biological Process: transcription, DNA-dependent; in utero embryonic development; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; negative regulation of transcription factor activity; proteasomal ubiquitin-independent protein catabolic process; protein ubiquitination; regulation of epidermal cell differentiation
NCBI Summary:	This gene encodes a protein containing KELCH-1 like domains, as well as a BTB/POZ domain. Kelch-like ECH-associated protein 1 interacts with NF-E2-related factor 2 in a redox-sensitive manner and the dissociation of the proteins in the cytoplasm is followed by transportation of NF-E2-related factor 2 to the nucleus. This interaction results in the expression of the catalytic subunit of gamma-glutamylcysteine synthetase. Two alternatively spliced transcript variants encoding the same isoform have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	Q14145
NCBI GenInfo Identifier:	146345444
NCBI Gene ID:	9817
NCBI Accession:	Q14145.2
UniProt Related Accession:	Q14145
Molecular Weight:
NCBI Full Name:	Kelch-like ECH-associated protein 1
NCBI Synonym Full Names:	kelch like ECH associated protein 1
NCBI Official Symbol:	KEAP1Â Â
NCBI Official Synonym Symbols:	INrf2; KLHL19Â Â
NCBI Protein Information:	kelch-like ECH-associated protein 1
UniProt Protein Name:	Kelch-like ECH-associated protein 1
UniProt Synonym Protein Names:	Cytosolic inhibitor of Nrf2; INrf2; Kelch-like protein 19
Protein Family:	Kelch-like ECH-associated protein
UniProt Gene Name:	KEAP1Â Â
UniProt Entry Name:	KEAP1_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 Âµl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 Âµl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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.