The Human Nuclear Factor Kappa B (NFkB) ELISA kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of NFkB in human tissue homogenates, cell lysates and other biological fluids.
Detection Range:0.156-10ng/ml
Sensitivity:<0.054ng/ml
Precision:Intra-Assay: CV<10%Inter-Assay: CV<12%
Test Principle:The microtiter plate provided in this kit has been pre-coated with an antibody specific to NFkB. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to 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 NFkB, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulfuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of NFkB in the sample is then determined by comparing the O.D. of the sample to the standard curve.
Storage and Stability:Store *027187A, *027187B, *027187D and *027187E at -20°C. Store all the other components at 4°C. Unused kit is stable for 6 months. Once kit components are opened, it is highly recommended to use remaining reagents within 1 month provided this is within the expiration date of the kit. For maximum recovery of product, centrifuge the original vials after thawing and prior to removing the cap.
Materials Required But Not Supplied:1. Microplate reader with 450 ± 10nm filter
2. Precision single or multi-channel pipettes and disposable tips
3. Eppendorf Tubes for diluting samples
4. Deionized or distilled water
5. Absorbent paper for blotting the microtiter plate
6. Container for Wash Solution
Sample Preparation and Storage:Tissue Homogenates:The preparation of tissue homogenates will vary depending upon tissue type
1. Rinse tissue thoroughly in ice-cold PBS (0.02M pH 7.0-7.2) to remove excess blood and weigh before homogenization. 2. Mince the tissue to small pieces and homogenize in fresh lysis buffer (1ml lysis buffer per 20-50mg tissue sample) using a glass homogenizer on ice (micro tissue grinders may also be used). Note: Use lysis buffer composition that is appropriate for the sample; choice depends on the sub-cellular localization of the target protein. Our Cat. #366747 (Tissue and Cell Lysis Buffer Set for ELISA) can be purchased separately
3. Subject the resulting suspension to sonication using an ultrasonic cell disrupter until a clarified mixture is obtained
4. Centrifuge the homogenate for 5 minutes at 10,000 x g. Collect the supernatant and assay immediately or aliquot and store at -20°C or lower.
Cell Lysates:Cells must be lysed before assaying according to the following directions. 1. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly). 2. Wash cells three times in cold PBS. 3. Resuspend cells in PBS (1×) and subject the cells to ultrasonication 4X (or freeze cells at -20°C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle 3X.) 4. Centrifuge at 1500×g for 10 minutes at 2-8°C to remove cellular debris.
Cell Culture Supernatants and Other Biological Fluids:Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquots at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
Note:1. Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C (≤1 month) or -80°C (≤2 months) to avoid loss of bioactivity and contamination
2. When performing the assay, bring samples to RT.
Assay Procedure Summary:1. Prepare all reagents, samples and standards
2. Add 100ul standard or sample to each well. Incubate 1 hour at 37°C
3. Aspirate and add 100ul prepared Detection Reagent A. Incubate 1 hour at 37°C
4. Aspirate and wash 3 times
5. Add 100ul prepared Detection Reagent B. Incubate 30 minutes at 37°C
6. Aspirate and wash 5 times
7. Add 90ul TMB Substrate. Incubate 10-20 minutes at 37°C
8. Add 50ul Stop Solution. Read at 450nm immediately.
