NFkB ELISA Kit

Catalog No. ABIN416230

Product Details NFkB ELISA Kit

Target: NFkB (Nuclear Factor kappa B (NFkB))

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.31 ng/mL - 20 ng/mL

Minimum Detection Limit: 0.31 ng/mL

Application: ELISA

Purpose: The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of NFkB in Tissue Homogenate,Cell Lysate,Biological Fluids

Sample Type: Cell Culture Supernatant, Cell Lysate, Tissue Homogenate

Analytical Method: Quantitative

Specificity:

This assay has high sensitivity and excellent specificity for detection of Nuclear Factor Kappa B (NFkB).
No significant cross-reactivity or interference between Nuclear Factor Kappa B (NFkB) and analogues was observed.

Cross-Reactivity (Details): No significant cross-reactivity or interference between Nuclear Factor Kappa B (NFkB) and analogues was observed.

Sensitivity: 0.114 ng/mL

Components:

• Pre-coated, ready to use 96-well strip plate, flat buttom
• Plate sealer for 96 wells
• Reference Standard
• Standard Diluent
• Detection Reagent A
• Detection Reagent B
• Assay Diluent A
• Assay Diluent B
• Reagent Diluent (if Detection Reagent is lyophilized)
• TMB Substrate
• Stop Solution
• Wash Buffer (30 x concentrate)
• Instruction manual

Application Details

Application Notes:

• Limited by the current condition and scientific technology, we cannot completely conduct the comprehensive identification and analysis on the raw material provided by suppliers. So there might be some qualitative and technical risks to use the kit.
• The final experimental results will be closely related to validity of the products, operation skills of the end users and the experimental environments. Please make sure that sufficient samples are available.
• Kits from different batches may be a little different in detection range, sensitivity and color developing time.
• Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.
• Protect all reagents from strong light during storage and incubation. All the bottle caps of reagents should be covered tightly to prevent the evaporation and contamination of microorganism.
• There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results. Do not remove microtiter plate from the storage bag until needed.
• Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting for the plate reader may lead to incorrect results. A microplate plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 O.D. or greater at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. Please read the instruction carefully and adjust the instrument prior to the experiment.
• Even the same operator might get different results in two separate experiments. In order to get better reproducible results, the operation of every step in the assay should be controlled. Furthermore, a preliminary experiment before assay for each batch is recommended.
• Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipments. Intra-assay variance among kits from different batches might arise from above factors, too.
• Kits from different manufacturers for the same item might produce different results, since we have not compared our products with other manufacturers.

Comment:

Information on standard material:
The standard might be recombinant protein or natural protein, that will depend on the specific kit. Moreover, the expression system is E.coli or yeast or mammal cell. There is 0.05% proclin 300 in the standard as preservative.

Information on reagents:
The stop solution used in the kit is sulfuric acid with concentration of 1 mol/L. And the wash solution is TBS. The standard diluent contains 0.02 % sodium azide, assay diluent A and assay diluent B contain 0.01% sodium azide. Some kits can contain is BSA in them.

Information on antibodies:
The provided antibodies and their host vary in different kits.

Sample Volume: 100 μL

Assay Time: 3 h

Plate: Pre-coated

Protocol: The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Nuclear Factor Kappa B (NFkB). Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Nuclear Factor Kappa B (NFkB). Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Nuclear Factor Kappa B (NFkB), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of Nuclear Factor Kappa B (NFkB) in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reagent Preparation:

1. Bring all kit components and samples to room temperature (18-25 °C) before use. If the kit will not be used up in one time, please only take out strips and reagents for present experiment, and leave the remaining strips and reagents in required condition.
2. Standard - Reconstitute the Standard with 1.0 mL of Standard Diluent, keep for 10 minutes at room temperature, shake gently (not to foam). The concentration of the standard in the stock solution is 40 ng/mL. Firstly dilute the stock solution to 20 ng/mL and the diluted standard serves as the highest standard (20 ng/mL). Then prepare 7 tubes containing 0.5 mL Standard Diluent and use the diluted standard to produce a double dilution series. Mix each tube thoroughly before the next transfer. Set up 7 points of diluted standard such as 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.312 ng/mL, and the last microcentrifuge tube with Standard Diluent is the blank as 0 ng/mL.
3. Detection Reagent A and Detection Reagent B - If lyophilized reconstitute the Detection Reagent A with 150μL of Reagent Diluent, keep for 10 minutes at room temperature, shake gently (not to foam). Briefly spin or centrifuge the stock Detection A and Detection B before use. Dilute them to the working concentration 100-fold with Assay Diluent A and B, respectively.
4. Wash Solution - Dilute 20 mL of Wash Solution concentrate (30x) with 580 mL of deionized or distilled water to prepare 600 mL of Wash Solution (1x).
5. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.

Note:

1. Making serial dilution in the wells directly is not permitted.
2. Prepare standards within 15 minutes before assay. Please do not dissolve the reagents at 37 °C directly.
3. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals are completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10μL for one pipetting.
4. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
5. If crystals have formed in the Wash Solution concentrate (30x), warm to room temperature and mix gently until the crystals are completely dissolved.
6. Contaminated water or container for reagent preparation will influence the detection result.

Assay Precision:

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Nuclear Factor Kappa B (NFkB) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Nuclear Factor Kappa B (NFkB) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Restrictions: For Research Use only

Handling

Precaution of Use: The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

Handling Advice: The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5 % within the expiration date under appropriate storage condition.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Storage: 4 °C

Storage Comment:

• For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20°C upon receipt while the others should be at 4°C.
• For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.
  Note: It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit.
• For ELISA kit, 1 day storage at 37°C can be considered as 2 months at 4°C, which means 3 days at 37°C equaling 6 months at 4°C.

Expiry Date: 6 months

References for NFkB ELISA Kit (ABIN416230)

Madi, Choucry, El-Marasy, Meselhy, El-Kashoury: "UPLC-Orbitrap HRMS metabolic profiling of Cymbopogon citratus cultivated in Egypt; neuroprotective effect against AlCl3-induced neurotoxicity in rats." in: Journal of ethnopharmacology, Vol. 259, pp. 112930, (2020) (PubMed).

Lv, Li, Hu, Wang, Wu, Li et al.: "The shear wave elastic modulus and the increased nuclear factor kappa B (NF-kB/p65) and cyclooxygenase-2 (COX-2) expression in the area of myofascial trigger points activated in a rat model by blunt ..." in: Journal of biomechanics, Vol. 66, pp. 44-50, (2019) (PubMed).

Zaafan, Haridy, Abdelhamid: "Amitriptyline attenuates bleomycin-induced pulmonary fibrosis: modulation of the expression of NF-κβ, iNOS, and Nrf2." in: Naunyn-Schmiedeberg's archives of pharmacology, Vol. 392, Issue 3, pp. 279-286, (2019) (PubMed).

Nahm, Nahm, Han, Gil, Choi, Lee: "Increased cerebral nuclear factor kappa B in a complex regional pain syndrome rat model: possible relationship between peripheral injury and the brain." in: Journal of pain research, Vol. 12, pp. 909-914, (2019) (PubMed).

George, Esmat, Tadros, El-Demerdash: "In vivo cellular and molecular gastroprotective mechanisms of chrysin; Emphasis on oxidative stress, inflammation and angiogenesis." in: European journal of pharmacology, Vol. 818, pp. 486-498, (2018) (PubMed).

Baluchnejadmojarad, Zeinali, Roghani: "Scutellarin alleviates lipopolysaccharide-induced cognitive deficits in the rat: Insights into underlying mechanisms." in: International immunopharmacology, Vol. 54, pp. 311-319, (2018) (PubMed).

Khajevand-Khazaei, Ziaee, Motevalizadeh, Rohani, Afshin-Majd, Baluchnejadmojarad, Roghani: "Naringenin ameliorates learning and memory impairment following systemic lipopolysaccharide challenge in the rat." in: European journal of pharmacology, Vol. 826, pp. 114-122, (2018) (PubMed).

Fayed, El-Naga, Akool, El-Demerdash: "The potential antifibrotic impact of apocynin and alpha-lipoic acid in concanavalin A-induced liver fibrosis in rats: Role of NADPH oxidases 1 and 4." in: Drug discoveries & therapeutics, Vol. 12, Issue 2, pp. 58-67, (2018) (PubMed).

Afshin-Majd, Bashiri, Kiasalari, Baluchnejadmojarad, Sedaghat, Roghani: "Acetyl-l-carnitine protects dopaminergic nigrostriatal pathway in 6-hydroxydopamine-induced model of Parkinson's disease in the rat." in: Biomedicine & pharmacotherapy, Vol. 89, pp. 1-9, (2017) (PubMed).

Kiasalari, Heydarifard, Khalili, Afshin-Majd, Baluchnejadmojarad, Zahedi, Sanaierad, Roghani: "Ellagic acid ameliorates learning and memory deficits in a rat model of Alzheimer's disease: an exploration of underlying mechanisms." in: Psychopharmacology, Vol. 234, Issue 12, pp. 1841-1852, (2017) (PubMed).

Sedaghat, Taab, Kiasalari, Afshin-Majd, Baluchnejadmojarad, Roghani: "Berberine ameliorates intrahippocampal kainate-induced status epilepticus and consequent epileptogenic process in the rat: Underlying mechanisms." in: Biomedicine & pharmacotherapy, Vol. 87, pp. 200-208, (2017) (PubMed).

Diling, Tianqiao, Jian, Chaoqun, Ou, Yizhen: "Docking Studies and Biological Evaluation of a Potential β-Secretase Inhibitor of 3-Hydroxyhericenone F from Hericium erinaceus." in: Frontiers in pharmacology, Vol. 8, pp. 219, (2017) (PubMed).

Diling, Chaoqun, Jian, Jian, Jiyan, Yizhen, Guoxiao: "Immunomodulatory Activities of a Fungal Protein Extracted from Hericium erinaceus through Regulating the Gut Microbiota." in: Frontiers in immunology, Vol. 8, pp. 666, (2017) (PubMed).

Abd El Motteleb, Ibrahim, Elshazly: "Sildenafil protects against bile duct ligation induced hepatic fibrosis in rats: Potential role for silent information regulator 1 (SIRT1)." in: Toxicology and applied pharmacology, Vol. 335, pp. 64-71, (2017) (PubMed).

Kiasalari, Khalili, Baluchnejadmojarad, Roghani: "Protective Effect of Oral Hesperetin Against Unilateral Striatal 6-Hydroxydopamine Damage in the Rat." in: Neurochemical research, Vol. 41, Issue 5, pp. 1065-72, (2016) (PubMed).

El-Agroudy, El-Naga, El-Razeq, El-Demerdash: "Forskolin, a hedgehog signalling inhibitor, attenuates carbon tetrachloride-induced liver fibrosis in rats." in: British journal of pharmacology, Vol. 173, Issue 22, pp. 3248-3260, (2016) (PubMed).

Kiasalari, Rahmani, Mahmoudi, Baluchnejadmojarad, Roghani: "Diosgenin ameliorates development of neuropathic pain in diabetic rats: Involvement of oxidative stress and inflammation." in: Biomedicine & pharmacotherapy, Vol. 86, pp. 654-661, (2016) (PubMed).

Ahshin-Majd, Zamani, Kiamari, Kiasalari, Baluchnejadmojarad, Roghani: "Carnosine ameliorates cognitive deficits in streptozotocin-induced diabetic rats: Possible involved mechanisms." in: Peptides, Vol. 86, pp. 102-111, (2016) (PubMed).

Baluchnejadmojarad, Kiasalari, Afshin-Majd, Ghasemi, Roghani: "S-allyl cysteine ameliorates cognitive deficits in streptozotocin-diabetic rats via suppression of oxidative stress, inflammation, and acetylcholinesterase." in: European journal of pharmacology, Vol. 794, pp. 69-76, (2016) (PubMed).

Zarezadeh, Baluchnejadmojarad, Kiasalari, Afshin-Majd, Roghani: "Garlic active constituent s-allyl cysteine protects against lipopolysaccharide-induced cognitive deficits in the rat: Possible involved mechanisms." in: European journal of pharmacology, Vol. 795, pp. 13-21, (2016) (PubMed).

Target Details for NFkB

Target: NFkB (Nuclear Factor kappa B (NFkB))

Alternative Name: NFkB (NFkB Products)

Synonyms: EBP-1 ELISA Kit, KBF1 ELISA Kit, NF-kB1 ELISA Kit, NF-kappa-B ELISA Kit, NF-kappaB ELISA Kit, NFKB-p105 ELISA Kit, NFKB-p50 ELISA Kit, NFkappaB ELISA Kit, p105 ELISA Kit, p50 ELISA Kit, NF-kB ELISA Kit, NF-KB1 ELISA Kit, NF-kappaB1 ELISA Kit, p50/p105 ELISA Kit, nuclear factor kappa B subunit 1 ELISA Kit, nuclear factor of kappa light polypeptide gene enhancer in B cells 1, p105 ELISA Kit, NFKB1 ELISA Kit, Nfkb1 ELISA Kit

UniProt: Q63369

Pathways: Ubiquitin Proteasome Pathway, S100 Proteins