NFkB (Nuclear Factor Kappa B) BioAssay(TM) ELISA Kit (Human)

Artikelname:	NFkB (Nuclear Factor Kappa B) BioAssay(TM) ELISA Kit (Human)
Artikelnummer:	USB-027187
Hersteller Artikelnummer:	027187
Alternativnummer:	USB-027187-48,USB-027187-96
Hersteller:	US Biological
Kategorie:	Kits/Assays

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. Kit Components: *027187A: Microtiter Plate, 1x96 wells, Pre-coated, ready to use *027187B: Standard, 2x1vial 027187C: Standard Diluent, 1x20ml *027187D: Detection Reagent A, 1x120ul *027187E: Detection Reagent B, 1x120ul 027187F: Assay Diluent A, 1x12ml 027187G: Assay Diluent B, 1x12ml 027187H: TMB Substrate, 1x9ml 027187K: Stop Solution, 1x6ml 027187L: Wash Buffer, 30X, 1x20ml Storage and Stability: Store *027187A, *027187B, *027187D and *027187E at -20C. Store all the other components at 4C. 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 -20C 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 -20C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle 3X.) 4. Centrifuge at 1500*g for 10 minutes at 2-8C 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 -20C or -80C. Avoid repeated freeze/thaw cycles. Note: 1. Samples to be used within 5 days may be stored at 4C, otherwise samples must be sto

NCBI:	19838
UniProt:	P19838