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  • 產品名稱：p65 ELISA Kit(NuclearFactor-KBP65)

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  • 產品廠商：國內供應3
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p65 ELISA Kit(NuclearFactor-KBP65)
詳情介紹：
Purpose This immunoassay kit allows for the in vitro quantitative determination of human NF- kappa B p65 concentrations in serum, plasma, tissue homogenates and other biological fluids.
Sample Type Serum, Plasma, Tissue Homogenate, Biological Fluids
Analytical Method Quantitative
Detection Method Colorimetric
Specificity This assay recognizes recombinant and natural human NF- kappa B p65.
Cross-Reactivity (Details) No significant cross-reactivity or interference was observed.
Sensitivity The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.
Characteristics Homo sapiens,Human,Transcription factor p65,Nuclear factor NF-kappa-B p65 subunit,Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3,RELA,NFKB3
Components Reagent (Quantity ): Assay plate (1), Standard (2), Sample Diluent (1 × 20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A (1 × 120μl), Detection Reagent B (1 × 120μl), Wash Buffer (25 x concentrate) (1 × 30ml), Substrate (1x10ml), Stop Solution (1x10ml), Plate sealer for 96 wells (5), Instruction (1)
Material not included Microplate reader. Pipettes and pipette tips. EP tube Deionized or distilled water.
Alternative Name RELA (NFkBP65 ELISA Kit Abstract)
Background Rel/nuclear factor- kappa B (NF- kappa B) is a dimeric transcription factor that plays important roles in the control of growth, differentiation, and apoptosis. It is also involved in immune and adaptive responses to changes in cellular redox balance [6 – 8]. Rel/NF- kappa B consists of homodimers and heterodimers formed by several subunits: NF- kappa B1 (p50/p105), NF- kappa B2 (p52/100), Rel A (p65), Rel B, and c-Rel proteins. These subunits have a high level of sequence homology within the NH2-terminal 300 amino acids in the Rel homology domain. The inactive form of NF- kappa B is localized in the cytoplasm and consists of three subunits: DNA-binding p50 and p65 subunits and an inhibitory subunit, called I kappa B, which is bound to p65. I kappa B masks the nuclear localization sequence and its release initiates activation of NF- kappa B and its subsequent translocation to the nucleus, where it can bind to target sites in DNA. Activation of NF- kappa B results in the induction of a large number of genes that influence cellular proliferation, inflammation, and cellular adhesion. A number of critical genes that are regulated by NF- kappa B include: anti-apoptotic genes (cIAP, survivin, Bcl2, and BClxL), cell cycle-regulatory genes (cyclin D1), genes encoding adhesion molecules, chemokines, inflammatory cytokines, and genes involved in tumor metastases (matrix metalloproteinase-9 [MMP-9], cyclooxygenase-2 [COX-2], nitric oxide synthase-2 [NOS-2], and vascular endothelial growth factor [VEGF]). Inappropriate regulation of NF- kappa B and its dependent genes has been associated with various pathologic conditions including toxic/septic shock, graft-versus-host disease, acute inflammatory conditions, acute-phase response, viral replication, radiation damage, atherosclerosis, and cancer.
Gene ID 3106
Pathways NF-kappaB Signaling, RTK Signaling, TCR Signaling, TLR Signaling, Fc-epsilon Receptor Signaling Pathway, Neurotrophin Signaling Pathway, Activation of Innate immune Response, Cellular Response to Molecule of Bacterial Origin, Hepatitis C, Toll-Like Receptors Cascades
Sample Volume 100 μL
Plate Pre-coated
Protocol The microtiter plate provided in this kit has been pre-coated with an antibody specific to NF- kappa B p65. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for NF- kappa B p65. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain NF- kappa B p65, biotin-conjugated antibody and enzyme-conjugated Avidin will 2 exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm 2 nm. The concentration of NF- kappa B p65 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Reagent Preparation

Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 2000 ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (2000 ng/mL). The Sample Diluent serves as the zero standard (0 ng/mL). Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.
Sample Collection Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8 °C within 30 minutes of collection. Store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Note: Citrate plasma has not been validated for use in this assay.
Assay Procedure

Allow all reagents to reach room temperature. Arrange and label required number of strips.
1. Prepare all reagents, working standards and samples as directed in the previous sections.
2. Add 100 uL of Standard, Control, or sample* per well. Cover with the adhesive strip. Incubate for 2 hours at 37 °C.
3. Remove the liquid of each well, don’t wash.
4. Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37°C. Detection 3 Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
5. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
6. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37 °C.
7. Repeat the aspiration/wash as in step
5. 8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Important Note:1. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
2. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
3. Duplication of all standards and specimens, although not required, is recommended.
4. When mixing or reconstituting protein solutions, always avoid foaming.
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
6. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
Calculation of Results

Construct a standard curve by plotting the mean absorbance value for each TAFI standard versus the corresponding concentration of TAFI in %. A standard curve should be generated each time the assay is performed. Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data 4 may be linearized by plotting the log of the TAFI concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Restrictions For Research Use only
Handling Advice 1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
Storage 4 °C/-20 °C
Storage Comment The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.